JP5154724B2 - Mutant luciferase - Google Patents

Abstract

A recombinant protein having luciferase activity and at least 60 % similarity to a wild-type luciferase wherein in the sequence of the enzyme, the amino acid residue corresponding to residue 357 in <i>Photinus pyralis</i> luciferase is mutated as compared to the corresponding wild-type luciferase, such that the luciferase enzyme is able to emit light at a different wavelength as compared to the corresponding wild-type luciferase and/or has enhanced thermostability as compared to the corresponding wild-type luciferase. In general, the residue corresponding to 357 in Photinus pyralis luciferase is changed from an acidic amino acid to a non-acidic amino acid and preferably an uncharged polar amino acid such as tyrosine. Mutant luciferases in accordance with the invention can produce a large (50nm) wavelength shift in emitted light and have good thermostability. The resultant colour shift can be reversed by addition of coenzyme A. These properties make the mutant particularly useful in a variety of assays.

Description

【００７１】
上記変異体を作製するための変異は、部位特異的変異法、（ＰＣＲ）またはコンビナトリアルカセット変異によって、プラスミドｐＥＴ２３上のルシフェラーゼ遺伝子に導入した。適切な変異を行うための、ＰＣＲ反応に加えられたオリゴヌクレオチドは以下に示す。
【００７２】
３５４および２１５位置での点変異の影響は追加的なものであることが以前に報告している。本発明は、３つ以上のこのような変異を組み合わせる可能性を提供して、大きなカラーシフトを有する変異型酵素中における高い熱安定性を提供する。
【００７３】
本発明のルシフェラーゼタンパク質は、シグナル手段としてルシフェラーゼ／ルシフェリン反応を使用するいかなる生物発光アッセイにおいても、有利に使用できよう。文献中で知られている多くのこのようなアッセイが存在する。したがって、このようなアッセイを行う目的で作製されたキット中このタンパク質が含まれよう。場合によってはルシフェリンおよび特定のアッセイを行うために必要とされる任意の他の試薬も含まれる。
【００７４】
ここで本発明を、例示として添付の図面を参照することによって詳細に記載する。
【００７５】
図１は、残存活性％対本発明によるいくつかの変異型酵素の４５℃でのインキュベーションの時間を示す対数グラフである。
【００７６】
図２は、Ｄ−ルシフェリンを有するクエン酸バッファー中で、ルシフェラーゼ酵素を発現する大腸菌細胞をインキュベートすることによって得られたスペクトルのピークを示す。この場合、使用した酵素は（ａ）組換え野生型Ｐｈｏｔｉｎｕｓ ｐｙｒａｌｉｓのルシフェラーゼ、（ｂ）Ｄ３５７Ｋ変異型、（ｃ）Ｄ３５７Ｎ変異型、（ｄ）Ｄ３５７Ｗ変異型、（ｅ）Ｄ３５７Ｉ変異型、（ｆ）Ｄ３５７Ｆ変異型、（ｇ）Ｄ３５７Ｙ変異型および（ｈ）二重変異型Ｅ３５４Ｉ＋Ｄ３５７Ｙである。
【００７７】
図３は、残存活性％対３つの変異型酵素Ｅ３５４Ｉ、Ｄ３５７Ｙおよび二重変異型（ＤＭ）Ｅ３５４Ｉ／Ｄ３５７Ｙの時間を示すグラフである。
【００７８】
図４は、（ａ）組換え野生型酵素および（ｂ）二重変異型（ＤＭ）Ｅ３５４Ｉ／Ｄ３５７Ｙの発光スペクトルを示す。
【００７９】
図５は、組換え野生型（◆）ｒ−ｗｔおよびＤ３５７Ｋ変異型酵素（■）の光量子発光の速度減衰を示すグラフである。
【００８０】
図６は分子モデル図を示し、ルシフェラーゼ酵素中の考えられるＣｏＡ結合ポケットを示す。
【００８１】
図７は、（ａ）ＬＢ上で成長させた、（ｂ）最小培地および酢酸ナトリウム上で成長させた、（ｃ）最小培地およびグルコース上で成長させた、変異体Ｐ．ｐｙｒａｌｉｓルシフェラーゼＤ３５７Ｙを発現する大腸菌細胞によって発せられたインビボでの生物発光スペクトルを示す。
【００８２】
図８は、（ａ）ＬＢ上で成長させた、（ｂ）最小培地および酢酸ナトリウム上で成長させた、（ｃ）最小培地およびグルコース上で成長させた、変異体Ｐ．ｐｙｒａｌｉｓのルシフェラーゼＥ３５４Ｋ／Ｄ３５７Ｍを発現する大腸菌細胞によって発せられたインビボでの生物発光スペクトルを示す。
【００８３】
図９は、変異体Ｐ．ｐｙｒａｌｉｓルシフェラーゼＤ３５７Ｙによって発せられた光のスペクトル分布に対するＣｏＡの影響を示すグラフである。
【００８４】
図１０は、変異体Ｐ．ｐｙｒａｌｉｓルシフェラーゼＤ３５７Ｙによって発せられた光のスペクトル分布に対するＣｏＡの影響の正規化したデータを示すグラフである。
【００８５】
図１１は、変異体Ｐ．ｐｙｒａｌｉｓルシフェラーゼＥ３５４Ｉ／Ｄ３５７Ｙによって発せられた光のスペクトル分布に対するＣｏＡの影響（図１１ａ）および正規化したデータ（図１１ｂ）を示すグラフである。
【００８６】
図１２は、合成ルシフェラーゼ遺伝子の構築において使用された制限部位の修飾を示す。
【００８７】
図１３は、ルシフェラーゼ遺伝子の合成において使用された構築体を示す。
【００８８】
図１４は、合成ルシフェラーゼ遺伝子のｃＤＮＡ配列（配列番号１）（リボソーム結合部位の一部を形成し、コードしていないヌクレオチド１〜８を含む）、および上向き矢印で示されたメチオニン残基で始まるエンコードされているアミノ酸配列（配列番号２）を示す。
【００８９】
図１５は、合成遺伝子によってエンコードされている変異体を含む、変異体の５０℃における熱安定性を示す。
【００９０】
実施例１
変異型ルシフェラーゼの同定および特徴付け
エラープロンＰＣＲ〔Ｍ．Ｆｒｏｍａｎｔ他、Ａｎａｌ．Ｂｉｏｃｈｅｍ．（１９９５）２２４、３４７−３５３〕を使用して作製した、ホタル（Ｐｈｏｔｉｎｕｓ ｐｙｒａｌｉｓ）ルシフェラーゼの２つのライブラリーを調製した。完全長のｌｕｃ遺伝子のエラープロンＰＣＲ産物からなる１つのライブラリーを、Ｔ７発現系ｐＥＴ２３ａにクローン化した（Ｎｏｖａｇｅｎ Ｉｎｃ．、Ｍａｄｉｓｏｎ、ＷＩ、Ｕ．Ｓ．Ａ）。１９９〜３５個のアミノ酸をカバーする、ｌｕｃ遺伝子の短い断片のエラープロンＰＣＲ産物からなる第２のライブラリーを、ベクターｐＢＳＫ（＋）中にクローン化した（Ｓｔｒａｔａｇｅｎｅ、Ｌａ Ｊｏｌｌａ、ＣＡ、Ｕ．Ｓ．Ａ）。
【００９１】
ｐＥＴ２３ａライブラリーを、大腸菌菌株ＢＬ２１（ＤＥ３）中で発現させた（大腸菌Ｂ Ｆ ｄｃｍ ｏｍｐｔ ｈｓｄＳ（ｒ_Ｂ ⁻ｍ_Ｂ ⁻）ｇａｌλ（ＤＥ３））。
【００９２】
ｐＢＳＫ（＋）ライブラリーを、ＨＢ１０１ 大腸菌細胞（ｓｕｐＥ４４ ａｒａｌ４ ｇａｌＫ２ ｌａｃＹ１ Δ（ｇｐｔ−ｐｒｏＡ）６２ ｒｐｓＬ２０（Ｓｔｒ^ｒ）ｘｙｌ−５ ｍｔｌ−１ ｒｅｃＡｌ３ Δ（ｍｒｃＣ−ｍｒｒ）ＨｓｄＳ⁻（ｒ⁻ｍ⁻））中で発現させた。ｐＥＴ２３ａおよびｐＢＳＫ（＋）は両方共にβ−ラクタマーゼの遺伝子を保有し、このプラスミドを有する大腸菌細胞にアンピシリン耐性を与える。
【００９３】
ＢＩＯＲＡＤ Ｅ．ｃｏｌｉ Ｐｕｌｓｅｒを使用するエレクトロポレーションによって、作製したライブラリーを用いて、大腸菌株細胞を形質転換させ、５０μｇ／ｍｌの濃度のアンピシリンを含むＬＢ寒天上で３７℃で一晩生育させた。細胞をナイロン膜（Ｏｓｍｏｎｉｃｓ、Ｍｉｎｎｅｔｏｎｋａ、Ｍｉｎｎｅｓｏｔａ、Ｕ．Ｓ．Ａ）に移し、ルシフェリン溶液（クエン酸ナトリウムバッファー１００ｍＭ、ｐＨ５．０中に５００μＭ Ｄ−ルシフェリン、カリウム塩、）をスプレーした。ＡｌｐｈＩｍａｇｅｒ（商標）１２００ＤｏｃｕｍｅｎｔａｔｉｏｎおよびＡｎａｌｙｓｉｓ Ｓｙｓｔｅｍ（Ｆｌｏｗｇｅｎ、Ｌｉｃｈｆｉｅｌｄ、Ｓｔａｆｆｏｒｄｓｈｉｒｅ、ＵＫ）を使用して、コロニーを観察した。これによって特定の時間で発せられる生物発光が取り込まれて、コロニーによって発せられる光の像を生み出す。発光の明るさを、ルシフェラーゼの熱安定性の指標として記録した。
【００９４】
次いで、熱安定性についてコロニーを選別した。発せられる光の明るさに基づいてコロニーを選択し、さらなる特徴付けのために単離した。いくつかの選別では、選別の前に大腸菌コロニーを４２℃で２時間インキュベートし、その結果熱安定性の変異体を選択することができた。最初の選別から単離したコロニーをナイロン膜の上に載せ、アンピシリンを含むＬＢ培地上で一晩生育させた。パッチをルシフェリン溶液でスプレーし、ＡｌｐｈＩｍａｇｅｒ（商標）中で観察した。この第２の選別は、ルシフェラーゼ活性のインビトロでの分析用のクローンを陽性に同定するのに役立った。可能性ある熱安定性酵素を発現する大腸菌のクローンを、ルシフェラーゼ活性および熱安定性についてインビトロでアッセイした。
【００９５】
室温で、Ｐｒｏｍｅｇａ Ｌｕｃｉｆｅｒａｓｅ Ａｓｓａｙ Ｓｙｓｔｅｍ（Ｐｒｏｍｅｇａ Ｃｏｒｐｏｒａｔｉｏｎ、Ｍａｄｉｓｏｎ、ＷＩ、Ｕ．Ｓ．Ａ）を使用して、ルシフェラーゼ活性についてインビトロでのアッセイを行った。
【００９６】
粗細胞抽出物１０μｌをＰｒｏｍｅｇａ Ｌｕｃｉｆｅｒａｓｅ Ａｓｓａｙ Ｃｏｃｋｔａｉｌ（１：２に希釈）に加えることによって、ルシフェラーゼ反応を開始させた。Ｂｉｏｔｒａｃｅ Ｍ３ 照度計を使用して、生じた生物発光を測定した。
【００９７】
Ｐｒｏｍｅｇａ ｔｅｃｈｎｉｃａｌ ｂｕｌｌｅｔｅｉｎ ｎｏ．１０１に記載されているように、粗細胞抽出物を調製した。細胞培養物溶解試薬（２５ｍＭ トリスリン酸塩 ｐＨ７．８、２ｍＭ ジチオスレイトール（ＤＴＴ）、２ｍＭ １，２−ジアミノシクロヘキサン−Ｎ，Ｎ，Ｎ’，Ｎ’−テトラ酢酸、１０％グリセロール、１％トリトン Ｘ−１００、１．２５ｍｇ／ｍｌ ｈｅｎ リソザイム）中で１０分間かけて室温で、一晩培養した大腸菌のアリコートを溶解させた。次いで粗溶解物をアッセイ前に氷上に保存した。
【００９８】
時間依存不活性化実験において、酵素の諸性質をさらに試験した。粗細胞抽出物のアリコート５０μｌを含むエッペンドルフチューブを、水浴中において所与の温度でインキュベートした。設定したいくつかのタイムポイントで、チューブを取り除きアッセイ前に氷上で冷却した。残存ルシフェラーゼ活性を、元の活性のパーセンテージとして表した。
【００９９】
残存活性％対インキュベーション時間の対数グラフをプロットし、ｔ_１／２値を計算するために使用した。Ｔ_１／２とは、酵素の、所与の温度でインキュベーション後の、その元の活性の５０％を失うのにかかる時間のことである。残存活性％に対する時間の対数グラフから、３７℃での粗抽出物中のＴ_１／２値（活性が元の活性の５０％に低下する時間）を決定した（示さず）。
【０１００】
上に述べたように決定した最も熱安定性のあるルシフェラーゼを発現する大腸菌クローンからのプラスミドＤＮＡを配列決定して、酵素の熱安定性を担う変異を決定した。
【０１０１】
ＱＩＡＧＥＮ ＱＩＡｐｒｅｐ Ｓｐｉｎ Ｍｉｎｉｐｒｅｐ Ｋｉｔ（ＱＩＡＧＥＮ Ｌｔｄ、Ｃｒａｗｌｅｙ、Ｗ．Ｓｕｓｓｅｘ、ＵＫ）、次にミクロ遠心分離を使用するためのプロトコル（ＱＩＡｐｒｅｐ Ｍｉｎｉｐｒｅｐ Ｈａｎｄｂｏｏｋ０４／９８）を使用して、プラスミドＤＮＡを作製した。
【０１０２】
ＤＮＡの配列決定はすべて、ＡＢＩ ＰＲＩＳＭ（商標）３７７ ＤＮＡ Ｓｅｑｕｅｎｃｅｒおよびジデオキシ鎖停止法〔Ｆ．Ｓａｎｇｅｒ他、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．Ｕ．Ｓ．Ａ ７４、（１９７７）５４６３−５４６７〕に基づくＡＢＩ ＰＲＩＳＭ（商標）ＢｉｇＤｙｅ（商標）Ｔｅｒｍｉｎａｔｏｒ Ｃｙｃｌｅ Ｓｅｑｕｅｎｃｅｒ Ｒｅａｄｙ Ｒｅａｃｔｉｏｎ Ｋｉｔ（Ｐｅｒｋｉｎ Ｅｌｍｅｒ Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）を使用して、Ｂａｂｒａｈａｍ Ｔｅｃｈ^ｎｉｘ、Ｃａｍｂｒｉｄｇｅ、ＵＫによって行った。
【０１０３】
この作業の結果、新規な変異体Ｄ３５７Ｙを特定した。
【０１０４】
ルシフェラーゼの結晶構造〔Ｅ．Ｃｏｎｔｉ他、Ｓｔｒｕｃｔｕｒｅ、４（１９９６）２８７−２９８〕は、位置３５７はタンパク質の表面に適合しており位置３５４に近く、熱安定性およびスペクトルの諸性質の両方に影響を及ぼす可能性があることを示す。これは、この領域は酵素の熱安定性の点で重要である可能性があることを示す。
【０１０５】
Ｄ３５７Ｙは非常に熱安定性のある変異体であって、最も熱安定性のあるルシフェラーゼであり、ただ１つのアミノ酸が変化している。
【０１０６】
実施例２
他の３５７変異体を作製するための部位特異的変異
３５７位置における異なる変異体を評価するために、Ｓｔｒａｔａｇｅｎｅ ＱｕｉｋＣｈａｎｇｅ（商標）Ｓｉｔｅ−Ｄｉｒｅｃｔｅｄ Ｍｕｔａｇｅｎｅｓｉｓ Ｋｉｔ（Ｓｔｒａｔａｇｅｎｅ、Ｌａ Ｊｏｌｌａ、ＣＡ、Ｕ．Ｓ．Ａ）を使用して、部位特異的変異を行った。プラスミドｐＰＷ６０１ａ Ｊ５４（ＰＪＷ、ＭｏＤ Ｒｅｐｏｒｔ、３／９６）を、すべての部位特異的変異において使用した。変異誘発反応のすべての産物を、大腸菌菌株ＸＬ１−Ｂｌｕｅ〔ｅ１４⁻（ｍｃｒＡ⁻）Δ（ｍｃｒＣＢ−ｈｓｄＳＭＲ−ｍｒｒ）１７１ ｅｎｄＡ１ ｓｕｐＥ４４ ｔｈｉ−１ ｇｙｒＡ９６ ｒｅｌＡｌ ｌａｃ ｒｅｃＢ ｒｅｃＪ ｓｂｃＣ ｕｍｕＣ：：Ｔｎ５（Ｋａｎ^ｒ）ｕｖｒＣ〔Ｆ’ｐｒｏＡＢ ｌａｃｌ^ｑｚΔｍ１５ Ｔｎ １０ （Ｔｅｔ^ｒ）Ａｍｙ Ｃａｍ^ｒ〕に形質転換した。Ｓｉｇｍａ−Ｇｅｎｏｓｙｓ Ｌｔｄ．、Ｃａｍｂｒｉｄｇｅ、ＵＫによってオリゴヌクレオチドプライマーを合成し、インテリジェントドーピングシステム〔Ａ．Ｒ Ａｒｋｉｎ他、Ｂｉｏ−ｔｅｃｈｎｏｌｏｇｙ、（１９９２）１０、２９７−３００、Ｗ、．Ｈｕｎｇ他、Ａｎａｌ．Ｂｉｏｃｈｅｍ．（１９９２）１０、４５４−４５７〕を使用して設計し、それぞれのアミノ酸置換に関する個々のプライマーを使用せずに、これを使用して縮重オリゴヌクレオチドプライマーを設計し、可能性ある変異体の群を作成した。
【０１０７】
このようにして、アミノ酸置換ルシフェラーゼ変異体のライブラリーを作成した。
【０１０８】
以下のオリゴヌクレオチド（およびその相補的なパートナー）を使用した。
【０１０９】
【化１】

【０１１０】
変異体のライブラリーを、前述のように熱安定性に関して選別した。以下の等式〔Ｓ．Ｃｌｉｍｉｅ他、Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．２６５（１９９０）１８７７６−１８７７９〕を使用して、選別したコロニーの数を計算した。
【０１１１】
Ｎ＝〔１ｎ（１−ｐ）〕／〔１ｎ（（ｎ−１）／ｎ〕
Ｎが選別されるコロニーの数の場合、ｎは標的位置における可能性あるコドンの数であり、Ｐは混合物中のそれぞれのコドンが、少なくとも１度の選別用にサンプル採取される確率である。この計算はＰ＝０．９５に基づいていた。部位特異的変異から得た変異体をルシフェラーゼ活性に関してアッセイし、時間依存熱不活性化実験において特徴付けした。
【０１１２】
このようにして望ましいとした変異体を、アンピシリンを含みＡ_２６０?０．５の４００ｍｌのＬＢ培地中で生育させた。次いでイソプロピルβ−チオガラクトシダーゼ（ＩＰＴＧ）を加え最終濃度１ｍＭにすることによって、ルシフェラーゼ発現を誘導した。細胞を３０℃で撹拌しながら３時間インキュベートし、次いで遠心分離によって採取した。生じた細胞のペレットを、Ｍａｘｉ−Ｓｃａｌｅ Ｂａｃｔｅｒｉａｌ Ｐｒｏｔｅｉｎ Ｅｘｔｒａｃｔｉｏｎに関するＢ−ＰＥＲ（商標）のプロトコルに従って、Ｂ−ＰＥＲ（商標）Ｐｒｏｔｅｉｎ Ｅｘｔｒａｃｔｉｏｎ Ｒｅａｇｅｎｔ、（Ｐｉｅｒｃｅ Ｃｈｅｍｉｃａｌ Ｃｏｍｐａｎｙ、Ｒｏｃｈｆｏｒｄ、Ｕ．Ｓ．Ａ）１０ｍｌ、ＤＴＴ１ｍＭ中に再懸濁させて、粗抽出物を作成した。再構築Ｓｉｇｍａ Ｐｒｏｔｅａｓｅ Ｉｎｈｉｂｉｔｏｒ Ｃｏｃｋｔａｉｌ、５００μｌ（Ｐｒｏｄｕｃｔ Ｎｏ．Ｐ８４６５、Ｓｉｇｍａ、Ｓａｉｎｔ Ｌｏｕｉｓ、Ｍｉｓｓｏｕｒｉ、Ｕ．Ｓ．Ａ）をＢ−ＰＥＲ（商標）溶液に加えて、内生プロテアーゼを阻害した。次いで細胞の溶解物を、３００００ｇで３０分間遠心分離した。
【０１１３】
粗抽出物の上澄みを、硫酸アンモニウムを用いて分画した。３０％と５５％の間の飽和状態で沈殿した分画は、ルシフェラーゼ活性を含んでいた。この物質を０．５ｍｌ Ｔｒｉｓ ＨＣｌ ｐＨ８．０、１ｍＭ ＤＴＴに再懸濁させ、熱不活性化およびスペクトルの実験用に使用した。
【０１１４】
変換Ｄ３５７Ｌ、Ｔ、Ｖ、Ｗ、Ｒ、Ｉ、Ｓ、Ｋ、ＮおよびＦを導入した。粗抽出物のインビトロでの熱不活性化実験において、これらの変異体を特徴付けた。
【０１１５】
部分精製した抽出物を、熱不活性化バッファー：飽和した硫酸アンモニウム１０％、１ｍＭジチオスレイトールおよび０．２％ＢＳＡを含む５０ｍＭ リン酸カリウムバッファー ｐＨ７．８中１１に１の比率で希釈した。
【０１１６】
タンパク質溶液のアリコート１１０μｌを、アッセイ前に設定した時間だけ４０℃または４５℃でインキュベートし、氷上で冷却した。次いでＰｒｏｍｅｇａ Ｌｕｃｉｆｅｒａｓｅ Ａｓｓａｙ Ｒｅａｇｅｎｔ（１：２に希釈）を使用して、実施例１に記載するようにルシフェラーゼ活性を測定した。
【０１１７】
その結果を表２および３、および図１中に示す。未精製抽出物のＴ１／２値を４０℃（表２）および４５℃（表３）において決定した。
【０１１８】
【表２】

【０１１９】
【表３】

【０１２０】
すべての置換が、組換え野生型と比較して、向上した熱安定性を示した。
【０１２１】
実施例３
発せられる光の波長の変化
位置３５７におけるアミノ酸置換が、インビトロで酵素によって発せられる光のスペクトルに影響を及ぼすことも観察した。実施例２に記載の大腸菌細胞培養物のアリコート（２５０μｌ）を３７℃で一晩生育させ、ミクロ遠心分離においてスピンダウンさせ、上澄みを取り除いた。Ｄ−ルシフェリン１５０μｌを含むクエン酸バッファー（ｐＨ５．０）中で、異なる変異型ルシフェラーゼを発現する細胞をインキュベートし、ＳＰＥＣＴＲＡｍａｘ（登録商標）Ｍｉｃｒｏｐｌａｔｅ Ｓｐｅｃｔｒｏｆｌｕｏｒｏｍｅｔｅｒ、（Ｍｏｌｅｃｕｌａｒ Ｄｅｖｉｃｅｓ Ｃｒｏｐ．Ｃａｌｉｆｏｒｎｉａ、Ｕ．Ｓ．Ａ）を使用して発光スペクトルを測定することによって、インビトロでの反応から発せられる光を分析した。変異体Ｄ３５７Ｙ、ＦおよびＩに関して、スペクトルのピークおよび波長の分布の大きな変化を観察した（図２（ａ）〜（ｇ））。これらの結果を以下の表４に要約する。
【０１２２】
さらに、インビボでの変異体の発光を、暗室で肉眼によって評価した。Ｄ３５７変異体は、その発光スペクトルにおいてさまざまな色を示した。特にＤ３５７Ｙ、ＦおよびＩは、発せられる光のより長い波長への大幅なシフトを示した。
【０１２３】
ある場合（たとえばＤ３５７Ｆ）、光の発光の色の変化はλ_ｍａｘのシフトによるだけでなく、可視光線の異なる波長からのスペクトルへの貢献の違いによるようである。
【０１２４】
【表４】

【０１２５】
ある種の３５７変異体のインビボでの発光のλ_ｍａｘの比較のために、組換え野生型（ｒ−ｗｔ）酵素を使用した。Ｄ３５７Ｙ、ＦおよびＩは、その波長最大値の大幅なシフトを示した。
【０１２６】
実施例４
ＣｏＡの存在または不在下での酵素の諸性質
実施例１に記載したようにして、硫酸アンモニウム沈殿によってＤ３５７Ｙを部分的に精製した。この部分的に精製したＤ３５７Ｙ酵素（５μｌ）をＰｒｏｍｅｇａ Ｌｕｃｉｆｅｒａｓｅ Ａｓｓａｙ Ｒｅａｇｅｎｔ１５０μｌと混合させた。他のアリコートをＣｏＡは存在しない（２５ｍＭ トリストリシン ｐＨ７．８、５．０ｍＭ ＭｇＳＯ_４、０．１ｍＭ ＥＤＴＡ、２ｍＭ ＤＴＴ、４７０μＭ Ｄ−ルシフェリン、５３０μＭ ＡＴＰ）同等のアッセイ用バッファーと混合させた。２つの反応物の発光スペクトルを測定した。これを図９および１０に示す。
【０１２７】
これらのスペクトルは、ＣｏＡの存在または不在下での生物発光の顕著な違い、およびλ_ｍａｘの大幅なシフトを示す。ＣｏＡのルシフェラーゼ反応のキネテイクスに対する影響を、ＲＬＵスケール（ＲＬＵ−相対的光単位）の違いによって見ることもできる。
【０１２８】
この発光の違いによって、ＣｏＡの存在を検出するためのアッセイにおいてこの酵素を使用する可能性が生じる。
【０１２９】
実施例５
二重変異体の作製および諸性質
実施例２に記載するようにして、部位特異的変異を使用して、熱安定性および発光の色に対するあらゆる累積的影響を調べるために、Ｅ３５４Ｉ＋Ｄ３５７Ｙの二重変異体を工作した。
【０１３０】
部分精製した二重変異体Ｅ３５４Ｉ＋Ｄ３５７Ｙを、熱不活性化バッファー：飽和した硫酸アンモニウム１０％、１ｍＭジチオスレイトールおよび０．２％ＢＳＡを含む５０ｍＭ リン酸カリウムバッファー ｐＨ７．８中１１に１の比率で希釈した。
【０１３１】
タンパク質溶液のアリコート１１０μｌを、アッセイ前に設定した時間だけ４０℃または４５℃でインキュベートし、氷上で冷却した。次いでＰｒｏｍｅｇａ Ｌｕｃｉｆｅｒａｓｅ Ａｓｓａｙ Ｒｅａｇｅｎｔ（１：２に希釈）を使用して、前述のようにルシフェラーゼ活性を測定した。
【０１３２】
二重変異体は、単一の変異体Ｅ３５４ＩおよびＤ３５７Ｙそれぞれと比較して、熱安定性の顕著な増大を示した（図３を参照）。部分精製した二重変異体の熱不活性化実験により、この変異体の熱安定性の増大を確認し、４５℃での不活性化において７．７分のｔ_１／２値を与えた。
【０１３３】
Ｅ３５４ＩおよびＤ３５７Ｙの個々の変異体より、この二重変異体は、より深い赤色の発光を示し、発光の色の添加性を示すことが示された。
【０１３４】
実施例３に記載のアッセイ用バッファーを使用して、組換え野生型および二重変異体Ｅ３５４Ｉ＋Ｄ３５７Ｙの粗抽出物の発光スペクトルも測定した。
【０１３５】
インビボで測定した発光スペクトルは、６１１ｎｍのλ_ｍａｘを与えた。しかしながら、このスペクトルは、波長の赤い領域からの発光に大いに貢献し、肉眼によって視覚化するとより深い赤色の外観となる。未精製抽出物の発光スペクトルは、ｒＷＴと比較して、スペクトル形状の明確な変化および４４ｎｍの波長シフトを示した（図４を参照）。
【０１３６】
インビボでの二重変異体の発光スペクトルは、発光（６１３ｎｍ）のピーク波長の帯幅をシャープにし、および５４０〜５６０ｎｍの範囲の光の波長の貢献の低下させる両方を示す。
【０１３７】
これらの変異の劇的な効果によって、この酵素のこの領域の生物発光の色への重要性が示される。
【０１３８】
実施例６
改善された光量子の流れ
変異体Ｄ３５７Ｋを発現している大腸菌細胞のインビボでの生物発光は、この位置の他の変異体と比較して、非常に鮮やかであることを観察した。時間における光量子の発光の速度を測定することができる光度計を使用して、この酵素のフラッシュキネテイクスを分析した。組換え野生型酵素または変異体Ｄ３５７を含む大腸菌無細胞抽出物のアリコートを、グローキネテイクスを助長するいかなる試薬（たとえば補酵素Ａ）も含まないルシフェラーゼアッセイ用カクテルに加えた。時間（１５秒）における光量子の発光の減衰の速度を両方の酵素について測定し、変異体Ｄ３５７Ｋは大幅に遅いことを観察した（図４）。言い換えれば、変異型酵素は、反応の少なくとも最初の１５秒間において、組換え野生型酵素よりも低い程度で阻害される反応キネテイクスを有する。
【０１３９】
実施例７
位置Ｅ３５４およびＤ３５７でのコンビナトリアルカセット変異
ステップ１
カセット変異用のプラスミドｐＰＷ６０１ａＪ５４の工作
２対の合成オリゴヌクレオチド（以下参照）を使用して、２つの新しい独特な制限部位をプラスミドｐＰＷ６０１ａ／Ｊ５４内のｌｕｃ遺伝子中に導入した。合計６個のサイレント変異を、６３塩基対隔ててこの遺伝子中のＳｐｅＩおよびＫｐｎＩ制限部位に導入した。これらの新しい部位を含むプラスミドを、ｐＰＷ６０１ａＪ５４ＳｐｅＩ／ＫｐｎＩと呼んだ。これらの制限部位の存在および近接性は、コンビナトリアルカセット変異を使用して、ホタルルシフェラーゼの主要配列中のアミノ酸の位置Ｅ３５４およびＤ３５７での、ランダムな置換の影響を探索することを可能にする。
【０１４０】
【化２】

【０１４１】
太線で強調したヌクレオチドはエンドヌクレアーゼ認識部位を形成し、および上側のケースのこれらはこの部位を作製するために必要なポイント変異の位置を形成する。
【０１４２】
ステップ２
カセットの設計およびライブラリーの構築
１対の合成オリゴヌクレオチドを合成した。このオリゴヌクレオチドを次いでアニールし、新しい制限部位で消化されたプラスミドｐＰＷ６０１ａＪ５４ＳｐｅＩ／ＫｐｎＩに直接連結することができる二本鎖カセットが作製された。このカセットは、天然に存在する２０個のアミノ酸の考えられるすべての組み合わせを、この一次配列中の位置Ｅ３５４およびＤ３５７において導入できるように設計された。
【０１４３】
【化３】

ループライブラリーオリゴヌクレオチドのそれぞれ２μｇを、５０ｍＭ Ｔｒｉｓ−ＨＣｌ ｐＨ７．４、２５ｍＭ ＮａＣｌを含むバッファー中に混合し、３分間１００℃に加熱した。次いでこの溶液をヒーティングブロック中で＜５０℃までゆっくりと冷却して、相補的な配列をアニールさせた。次いでアニールしたオリゴヌクレオチドをプラスミドｐＰＷ６０１ａＪ５４ＳｐｅＩ／ＫｐｎＩに連結させ、ＳｐｅＩおよびＫｐｎＩを用いてこれを消化した。次いでこの連結反応のアリコートを使用して、エレクトロポレーションを使用する大腸菌ＨＢ１０１細胞を形質転換した。エレクトロポレーションの後、形質転換した細胞をアンピシリン５０μｇ／ｍｌを含むＬＢ寒天プレート上に載せ、３７℃で一晩生育させた。翌日、８６９個のコロニーをプレートからランダムに採取し、これを使用してアンピシリンを含むＬＢ１ｍｌを９６スクエアウエルのプレート（Ｂｅｃｋｍａｎ）に接種した。このプレートをカバーし、振とうしながら３７℃で細胞を一晩生育させた。
【０１４４】
ステップ３
ランダムに選択したクローンのインビボでの選別
翌朝、定常期の一晩培養したもののアリコート５０μｌを、２つの透明プラスチック製丸底９６ウエルのミクロタイタープレート（Ｄｙｎｅｘ）に移した。１つのプレートをカバーし、加熱したブロック上で８分間インキュベートし（ブロック表面温度４５℃）、一方もう１つのブロックは３７℃に保った。次いで０．５ｍＭのＤ−ルシフェリンを含む１００ｍＭのクエン酸ナトリウムバッファー（ｐＨ５．０）５０μｌをウエルに加え、次いでこのプレートをｖｉｄｅｏ ｃａｍｅｒａ ｉｍａｇｅｒ ｃａｐｔｕｒｅ ｓｙｓｔｅｍ（Ａｌｐｈａ Ｉｍａｇｅｒ）に移すことによって、インビボでの両方のプレートからの細胞中のシフェラーゼ活性を室温で検出し記録した。加熱および調節された培養物によって発せられた光を１〜２分かけて積分し、その像を熱性紙フィルム上に記録した。
【０１４５】
フィルム上で記録した像の明るさの判定によって、最大の生物発光を示した７９個の培養物を、選別の第２ラウンド用に選択した。今回は培養物を、アッセイの前にヒーティングブロック上で１６分間インキュベートした。インビボでの熱安定性選別から選択した５５個のクローンの内、インビボでのスペクトル分析用に２５個を選択した。これらのクローンをＬＢ中において３７℃で一晩生育させ、翌朝一晩培養したもの２００μｌを翌朝遠心分離し、大腸菌細胞のペレットを０．５ｍＭのＤ−ルシフェリンを含む１００ｍＭのクエン酸ナトリウムバッファー（ｐＨ５．０）１５０μｌ中に再懸濁させた。次いで再懸濁させた細胞を白いプラスチック製のミクロタイタープレート中に置き、変異型ルシフェラーゼのそれぞれによって発せられるインビボでの生物発光のスペクトルを、Ｍｏｌｅｃｕｌａｒ Ｄｅｖｉｃｅｓ Ｓｐｅｃｔｒａｍａｘ、９６ウエルプレートの蛍光光度計を使用して分析した。それらの結果を以下の表１に要約する。
【０１４６】
ステップ４
変異の同定
インビボでの選別によって選択された２５個のクローンからプラスミドＤＮＡを調製し、遺伝子特異的配列決定プライマーを使用して配列決定した。一次配列中の位置３５４および３５７においてアミノ酸の変化を生じた変異を同定した。ある変異体は、位置Ｉ３５１にアミノ酸の置換をもたらした（表５）。
【０１４７】
【表５】

【０１４８】
いくつかの変異型ルシフェラーゼを熱安定性のインビボアッセイから選択した。これらのルシフェラーゼの大部分は、また発せられる光のインビボスペクトルにおいて大きな変化を示し、多くは光の長い波長（＞５８０ｎｍ）からのより大きな貢献を示した。いくつかのスペクトルは、６１０〜６１４ｎｍというただ１つのピーク周辺で非常に狭い帯幅を示す。
【０１４９】
Ｅ３５４およびＤ３５７のそれぞれ疎水性および芳香族アミノ酸での置き換え（たとえばＥ３５４Ｖ、Ｄ３５７Ｙ）は、インビボでのスペクトルの大きく変化をもたらし、６１２ｎｍ周辺で、狭い帯幅の単位ピークを示す。
【０１５０】
実施例８
熱安定性に関するインビボ選別
選択したクローンの無細胞抽出物を溶解によって調製し、それぞれの抽出物からのルシフェラーゼの熱安定性を、熱不活性化実験において測定した。それぞれの抽出物５０μｌをエッペンドルフチューブ中に置き、４、９および１６分間、４５℃に加熱した水浴中でインキュベートした。適切なタイムポイントにおいてアリコートを取り、残存ルシフェラーゼ活性を測定した。表６は、すべての変異型酵素および組換え野生型について残存活性％対時間を示す。
【０１５１】
これらの結果は、最も熱安定性であるルシフェラーゼは、位置３５７に芳香アミノ酸（Ｙ、ＦまたはＷ）、および位置３５４に大きな疎水性（ＶまたはＩ）、極性（Ｎ）または正に帯電している（ＫまたはＲ）アミノ酸を有するルシフェラーゼであることを示す。
【０１５２】
【表６】

【０１５３】
実施例９
発せられる光のインビボでのスペクトルに対する成長条件の影響
変異型ルシフェラーゼＥ３５４Ｋ＋Ｄ３５７Ｍ（前述の７のもの）を発現している大腸菌ＢＬ２１（ＤＥ３）細胞から発せられる、光のスペクトルに対する異なる炭素源の影響を調べた。
【０１５４】
細胞の培養物５０ｍｌをＬＢ培地上で対数期中期まで生育させ、次いで遠心分離によって採取した。細胞ペレットを１ｍｌの滅菌蒸留水中に再懸濁させ、次いでこの懸濁液のアリコート１００ｕｌを使用して、新鮮なＬＢ５ｍｌ、Ｍ９最小培地＋２ｍＭ酢酸ナトリウムまたはＭ９最小培地＋２ｍＭグルコースを２５ｍｌの滅菌チューブ中に接種した。この培養物を振とうしながら３７℃で成長させ続け、９０分（Ｄ３５７Ｙ）および１２０分（酵素７）後、細胞のアリコート２００ｕｌを取り遠心分離し、０．５ｍＭのＤ−ルシフェリンを含む１００ｍＭのクエン酸ナトリウムバッファー（ｐＨ５．０）１５０ｕｌ中に再懸濁させた。次いで再懸濁させた細胞をミクロタイタープレート中に置き、変異型ルシフェラーゼのそれぞれによって発せられる生物発光のインビボスペクトルを、Ｍｏｌｅｃｕｌａｒ Ｄｅｖｉｃｅｓ Ｓｐｅｃｔｒａｍａｘ、９６ウエルプレートの蛍光光度計を使用して分析した。それらの結果を図７および８に示す。
【０１５５】
これらの結果は、富化培地（ＬＢ）（図７ａ、８ａ）からの、唯一の炭素源としてアセテート（図７ｂ、８ｂ）またはグルコース（図７ｃ、８ｃ）のいずれかを有する限定最小培地への変化は、発せられる光がより長い波長にシフトと、より短い波長からの貢献の低下をもたらすことを示す。
【０１５６】
実施例１０
組換え野生型および変異型ルシフェラーゼの精製およびスペクトルの特徴付け
組換え野生型Ｐｈｏｔｉｎｕｓ ｐｙｒａｌｉｓ酵素および変異型ルシフェラーゼＤ３５７ＹおよびＥ３５４Ｉ＋Ｄ３５７Ｙを、生物発光反応のスペクトルに対する補因子補酵素Ａの影響を分析するために、均一になるまで精製した。３つのルシフェラーゼすべてを、嫌気性真菌Ｐｉｒｏｍｙｃｅｓ ｅｑｕｉｉからの１４３アミノ酸炭水化物結合モジュール（ＣＢＭ）への融合体として、精製した。このＣＢＭは、酸膨張性セルロースおよび可溶性炭水化物ガラクトマナンおよびグルコマナンに選択的に結合し、簡単な単一ステップのアフィニティ精製スキームのベースを形成することが示されている。
【０１５７】
ＣＢＭに融合したルシフェラーゼは、粗無細胞抽出物中でセルロースに結合することができ、これを洗浄し、次いで可溶性多糖を使用して選択的に溶離させた。このようにして精製した融合タンパク質をアッセイにおいて使用して、異なる量の補酵素Ａを含む反応中で発せられた光の波長を測定した。異なる量の補酵素Ａを含むアッセイ試薬１００μｌ（２５ｍＭ トリストリシン ｐＨ７．８、５．０ｍＭ ＭｇＳＯ_４、０．１ｍＭ ＥＤＴＡ、５３０μＭ ＡＴＰおよび４７０μＭ Ｄ−ルシフェリン）に、酵素（５μｌ）を加えた。図９〜１１は、精製したルシフェラーゼＤ３５７およびＥ３５４Ｉ＋Ｄ３５７Ｙによって発せられた光のスペクトルに対する、補酵素Ａの増大した濃度の影響を示す。
【０１５８】
真菌ＣＢＭのＣ末端に融合しホタルのルシフェラーゼを発現している大腸菌細胞によって発せられる、生物発光のスペクトルのインビボでのアッセイは、元のルシフェラーゼを発現している細胞といかなる顕著な違いも示さなかった。同様に、精製組換えルシフェラーゼの市販品（Ｐｒｏｍｅｇａ）によって発せられる、生物発光のスペクトルのインビトロアッセイは、融合タンパク質によって発せられたスペクトルと同一であった。
【０１５９】
したがって、観察した違いはＣｏＡの濃度に伴われたものである。補酵素Ａの濃度が増大すると、スペクトルの分布が変化し、ＣｏＡの最大濃度では、スペクトルは、６１０ｎｍでの明確なピークを有する５９０〜６３０ｎｍの領域の波長が主となる。スペクトルのシフトは二重変異体に関して最も顕著であり、波長６１０ｎｍという唯一のピーク周辺で帯幅の大幅な狭まりが存在する（図１１）。
【０１６０】
実施例１２
２１４Ｃ／３５４Ｋ／３５７Ｆを有するように変異させた、合成Ｐｈｏｔｉｎｕｓ ｐｙｒａｌｉｓルシフェラーゼの生産
前述の合成戦略を使用して、オリゴヌクレオチド対から合成ｌｕｃ遺伝子を設計し組み立てた。遺伝子配列を工作して、アミノ酸２１４Ｃ、３５４Ｋおよび３５７Ｆを有するルシフェラーゼを作製した。
【０１６１】
重複合成オリゴヌクレオチド２９対をＳｉｇｍａ−Ｇｅｎｏｓｙｓ Ｌｔｄによって合成し、ＰＡＧＥによって精製し、約５５０ｂｐの３つのアセンブリに連結させた（ＩＤＲＩＳ１、２および３、図１３）。次いでそれぞれのアセンブリをベクターｐＢＳＫ（＋）別々に連結させ、生じた構築体を使用して大腸菌ＸＬ１−Ｂｌｕｅ細胞を形質転換させた。組み立てた挿入体を含むクローンからプラスミドＤＮＡを作製し、配列決定してＯＲＦの適合度を確認した。アセンブリ中のｎ−１個のオリゴヌクレオチドの存在（オリゴ合成の副産物）によって、構築プロセスが複雑になった。ＤＮＡの配列決定によってＩＤＲＩＳ２の１つの正確なアセンブリを同定し、ＰＣＲを使用して、構築体の５’端に１つの塩基対欠失体を含むＩＤＲＩＳ３の１アセンブリを正しくした。ＩＤＲＩＳ１を含むプラスミドの、ＩＤＲＩＳ２および３との混合物を連結させることによって、完全なＯＲＦのアセンブリを得た。
【０１６２】
次いで連結したＤＮＡを使用して大腸菌ＸＬ１−Ｂｌｕｅ細胞を形質転換させ、インビボアッセイを使用して活性酵素を発現しているクローンを選択した。数個のクローンを選択し、配列決定して図１４に示す配列を有する合成ｌｕｃ遺伝子の存在および適合度を確認した。完全なＯＲＦをＩＤＲＩＳ（ＦＡ）と呼んだ。
【０１６３】
合成遺伝子をベクターｐＢＳＫ（＋）内のポリリンカー中のＢａｍＨ ＩとＳａｌ Ｉ部位の間に組立てた。この位置において、この遺伝子はアルファペプチドとはフレームが同じでなく、ｌａｃプロモーターからは相当な距離がある。しかしながら、充分なルシフェラーゼが生成されて、酵素の予備的特徴付けが可能である。Ｐｒｏｍｅｇａ溶解法を使用して一晩培養したものから、ＩＤＲＩＳ（ＦＡ）を発現している大腸菌ＸＬ１−Ｂｌｕｅ細胞の粗無細胞抽出物を作製した。
【０１６４】
次いで抽出物中の酵素の熱安定性を５０℃で２０分間試験し、熱安定性の変異体Ｅ３５４Ｉ＋Ｄ３５７Ｙと比較した。新しいコドンを最適化させた三重変異体は、変異体Ｅ３５４Ｉ＋Ｄ３５７Ｙよりも大幅に熱安定性であった（図１５）。
【図面の簡単な説明】
【図１】 残存活性％対本発明によるいくつかの変異型酵素の４５℃でのインキュベーションの時間を示す対数グラフである。
【図２（ａ）】 Ｄ−ルシフェリンを有するクエン酸バッファー中で、ルシフェラーゼ酵素を発現する大腸菌細胞をインキュベートすることによって得られたスペクトルのピークを示す。使用した酵素は組換え野生型Ｐｈｏｔｉｎｕｓ ｐｙｒａｌｉｓのルシフェラーゼである。
【図２（ｂ）】 Ｄ−ルシフェリンを有するクエン酸バッファー中で、ルシフェラーゼ酵素を発現する大腸菌細胞をインキュベートすることによって得られたスペクトルのピークを示す。使用した酵素はＤ３５７Ｋ変異型である。
【図２（ｃ）】 Ｄ−ルシフェリンを有するクエン酸バッファー中で、ルシフェラーゼ酵素を発現する大腸菌細胞をインキュベートすることによって得られたスペクトルのピークを示す。使用した酵素はＤ３５７Ｎ変異型である。
【図２（ｄ）】 Ｄ−ルシフェリンを有するクエン酸バッファー中で、ルシフェラーゼ酵素を発現する大腸菌細胞をインキュベートすることによって得られたスペクトルのピークを示す。使用した酵素はＤ３５７Ｗ変異型である。
【図２（ｅ）】 Ｄ−ルシフェリンを有するクエン酸バッファー中で、ルシフェラーゼ酵素を発現する大腸菌細胞をインキュベートすることによって得られたスペクトルのピークを示す。使用した酵素はＤ３５７Ｉ変異型である。
【図２（ｆ）】 Ｄ−ルシフェリンを有するクエン酸バッファー中で、ルシフェラーゼ酵素を発現する大腸菌細胞をインキュベートすることによって得られたスペクトルのピークを示す。使用した酵素はＤ３５７Ｆ変異型である。
【図２（ｇ）】 Ｄ−ルシフェリンを有するクエン酸バッファー中で、ルシフェラーゼ酵素を発現する大腸菌細胞をインキュベートすることによって得られたスペクトルのピークを示す。使用した酵素はＤ３５７Ｙ変異型である。
【図２（ｈ）】 Ｄ−ルシフェリンを有するクエン酸バッファー中で、ルシフェラーゼ酵素を発現する大腸菌細胞をインキュベートすることによって得られたスペクトルのピークを示す。使用した酵素は二重変異型Ｅ３５４Ｉ＋Ｄ３５７Ｙである。
【図３】 残存活性％対３つの変異型酵素Ｅ３５４Ｉ、Ｄ３５７Ｙおよび二重変異型（ＤＭ）Ｅ３５４Ｉ／Ｄ３５７Ｙの時間を示すグラフである。
【図４（ａ）】 組換え野生型酵素の発光スペクトルを示す。
【図４（ｂ）】 二重変異型（ＤＭ）Ｅ３５４Ｉ／Ｄ３５７Ｙの発光スペクトルを示す。
【図５】 組換え野生型（◆）ｒ−ｗｔおよびＤ３５７Ｋ変異型酵素（■）の光量子発光の速度減衰を示すグラフである。
【図６】 分子のモデル図を示し、ルシフェラーゼ酵素中の考えられるＣｏＡ結合ポケットを示す。
【図７ａ】 ＬＢ上で成長させた変異体Ｐ．ｐｙｒａｌｉｓのルシフェラーゼＤ３５７Ｙを発現する大腸菌細胞によって発せられたインビボでの生物発光スペクトルを示す。
【図７ｂ】 最小培地および酢酸ナトリウム上で成長させた変異体Ｐ．ｐｙｒａｌｉｓのルシフェラーゼＤ３５７Ｙを発現する大腸菌細胞によって発せられたインビボでの生物発光スペクトルを示す。
【図７ｃ】 最小培地およびグルコース上で成長させた変異体Ｐ．ｐｙｒａｌｉｓのルシフェラーゼＤ３５７Ｙを発現する大腸菌細胞によって発せられたインビボでの生物発光スペクトルを示す。
【図８（ａ）】 ＬＢ上で成長させた変異体Ｐ．ｐｙｒａｌｉｓのルシフェラーゼＥ３５４Ｋ／Ｄ３５７Ｍを発現する大腸菌細胞によって発せられたインビボでの生物発光スペクトルを示す。
【図８（ｂ）】 最小培地および酢酸ナトリウム上で成長させた変異体Ｐ．ｐｙｒａｌｉｓのルシフェラーゼＥ３５４Ｋ／Ｄ３５７Ｍを発現する大腸菌細胞によって発せられたインビボでの生物発光スペクトルを示す。
【図８（ｃ）】 最小培地およびグルコース上で成長させた変異体Ｐ．ｐｙｒａｌｉｓのルシフェラーゼＥ３５４Ｋ／Ｄ３５７Ｍを発現する大腸菌細胞によって発せられたインビボでの生物発光スペクトルを示す。
【図９】 変異体Ｐ．ｐｙｒａｌｉｓのルシフェラーゼＤ３５７Ｙによって発せられた光のスペクトル分布に対するＣｏＡの影響を示すグラフである。
【図１０】 変異体Ｐ．ｐｙｒａｌｉｓのルシフェラーゼＤ３５７Ｙによって発せられた光のスペクトル分布に対するＣｏＡの影響を示すグラフである。
【図１１（ａ）】 変異体Ｐ．ｐｙｒａｌｉｓのルシフェラーゼＥ３５４Ｉ／Ｄ３５７Ｙによって発せられた光のスペクトル分布に対するＣｏＡの影響を示すグラフである。
【図１１（ｂ）】 変異体Ｐ．ｐｙｒａｌｉｓのルシフェラーゼＥ３５４Ｉ／Ｄ３５７Ｙによって発せられた光のスペクトル分布に対するＣｏＡの影響および正規化したデータを示すグラフである。
【図１２】 合成ルシフェラーゼ遺伝子の構築において使用された制限部位の修飾を示す。
【図１３】 ルシフェラーゼ遺伝子の合成において使用する構築体を示す。
【図１４】 合成ルシフェラーゼ遺伝子のｃＤＮＡ配列（配列番号１）（リボソーム結合部位の一部を形成し、コードしていないヌクレオチド１〜８含む）、および上向き矢印で示されたメチオニン残基で始まるエンコードされているアミノ酸配列（配列番号２）を示す。
【図１５】 合成遺伝子によってエンコードされている変異体を含む、変異体の５０℃における熱安定性を示す。
【配列表】

[0001]
The present invention includes novel proteins, particularly mutant luciferase enzymes that exhibit characteristic properties compared to the corresponding wild-type enzymes, DNA encoding these proteins, the use of these enzymes in assays, and the like It relates to a test kit.
[0002]
Firefly luciferase contains ATP, Mg²⁺And catalyzes the oxidation of luciferin in the presence of molecular oxygen, resulting in light. This reaction has a quantitative yield of about 0.88. This luminescence leads to use in a wide range of illuminance assays where ATP levels are measured. Examples of such assays include those based on the descriptions in EP-B-6580515 and WO 96/02665, but many other assays are routinely used in the laboratory.
[0003]
Luciferase can be obtained directly from the insect body, in particular from beetles such as fireflies or fireflies. Specific species from which the luciferase is derived include the Japanese GENJI or KEIKE fireflies, Lucioluca cruciata and Luciola lateralis, the Eastern European firefly Luciola mingrelica, the North American firefly Photinus pyritis, and a cybo litral.
[0004]
However, many of the genes encoding these enzymes have been cloned and sequenced and can also be generated using recombinant DNA techniques. The recombinant DNA sequence encoding the enzyme is used to transform a microorganism, such as E. coli, which thus expresses the desired enzyme product.
[0005]
When used in laboratory assays, the color of light emitted by these enzymes is very similar. If the wavelength is changed, it may be useful for easier reading by a particular detector, or for use in systems where multiple reporters are needed, for example to monitor different events in the same sample. One way to distinguish reporter molecules is to use luciferase molecules that emit light at specific wavelengths. This can be achieved by using a reporter molecule comprising luciferases from different species of beetles or moths. However, using recombinant DNA technology to generate mutant luciferases is an alternative strategy, resulting in variations in signal wavelength. Examples of such mutants are provided in WO95 / 18853.
[0006]
Furthermore, the thermal stability of wild and recombinant luciferases is such that they lose activity very rapidly when they are exposed to temperatures above about 30 ° C, especially above 35 ° C. This instability causes problems when the enzyme is used or stored at high ambient temperatures or when the assay is performed under high temperature reaction conditions, for example to increase the reaction rate.
[0007]
Mutant luciferases with increased thermostability are known from EP-A-524448 and WO / 95/25798. The first of these describes a mutant luciferase having a mutation at position 217 in Japanese firefly luciferase, specifically a threonine residue is replaced with an isoleucine residue. The latter describes a mutant luciferase with a similarity of more than 60% to the luciferase from Photinus pyralis, Luciola mingrelica, Luciola cruciata or Luciola lateralis, but to the residue of Photinus pylalis 56 type 35 Corresponding amino acid residues are mutated other than glutamate, and in particular, mutates other than glutamate, aspartate, proline or glycine.
[0008]
Co-pending UK patent application no. 98234688.5 and international patent applications derived therefrom further describe such variants. In this case, proteins that have luciferase activity and that have at least 60% similarity to wild-type luciferases from luciferases such as Photinus pyralis, Lucio mininglica, Lucioluca luciata or Luciola lateralis enzymes are described. It contains mutations at various positions, particularly including:
[0009]
(A) an amino acid residue corresponding to residue 214 in the Lucinase of Photinus pyralis, residue 216 of a Lucifera mingrelica, Luciola cruciata or Luciola lateralis luciferase, or
(B) residue 232 in Photinus pyralis luciferase, an amino acid residue corresponding to residue 234 of Luciola mingrelica, Luciola cruciata or Luciola lateralis luciferase, or
(C) Amino acid residue corresponding to residue 295 in Lucinus pyralis luciferase, residue 297 of Lucio mininglica, Lucioluca cruciata or Luciola lateralis luciferase.
[0010]
Applicants can obtain a large shift in the wavelength of the emitted light and / or improve the thermal stability of the enzyme by mutating (or introducing) amino acids at different positions in the luciferase protein. I discovered that. In addition, in in vivo assays where glow kinetics are hindered, or in in vitro assays where no CoA or other “glow kinetics-inducing” compounds are present, the flow of emitted protons can be improved and the enzyme more compatible.
[0011]
The present invention provides a recombinant protein having luciferase activity and at least 60% similarity to wild-type luciferase. In the sequence of this enzyme, the amino acid residue corresponding to residue 357 in Photinus pyralis luciferase is mutated compared to the corresponding wild-type luciferase, so that the luciferase enzyme is conjugated with the corresponding wild-type luciferase and It can emit light of different wavelengths in comparison and / or has improved thermal stability compared to the corresponding wild type luciferase.
[0012]
Recombinant wild-type luciferase sequence Photinus pyralis the base portion can be formed of the present invention, Luciola mingrelica, Luciola cruciata or Luciola lateralis, Hotaria parvula, Pyrophorus plagiophthalamus Lampyris noctiluca, Pyrocoelia miyako, Photuris pennsylvanica or Phrixothrix (railroad -Worms Biochem.38 (1999) 8271-8279).
[0013]
Bioluminescent enzymes from species that can produce luminescence using the substrate D-luciferin (4,5-dihydro-2- [6-hydroxy-2-benzothiazolyl] -4-thiazolecarboxylic acid) are disclosed in the present invention. The basic part of the mutant enzyme can be formed.
[0014]
Photinus pyralis for certain wild-type luciferase sequence the basic parts of the recombinant can be formed of the present invention, Luciola mingrelica, Luciola cruciata or Luciola lateralis, Hotaria parvula, Pyrophorus plagiophthalamus Lampyris noctiluca, there is Pyrocoelia miyako and Photuris pennsylvanica .
[0015]
In particular, this luciferase is an enzyme obtained from the enzymes of Photinus pyralis, Lucio minarelica, Luciola cruciata or Luciola lateralis. In the Lucio mingrelica, Lucio cruciata, or Lucio la lateralis enzyme, the appropriate amino acid residue is at position 359 in the sequence.
[0016]
The sequences of all the various luciferases indicate that they are highly conserved with high similarity between them. This means that by examining the sequences, the corresponding regions between the enzyme sequences can be easily determined and the most similar regions can be detected, but if necessary, the corresponding regions between the various sequences Alternatively, to determine individual amino acids, see commercially available software (see, eg, “Bestfit” from the University of Wisconsin Genetics Computer Group; see Devereux et al. (1984) Nucleic Acid Research 12: 387-395). it can. Alternatively or additionally, the sequence and numbering of this enzyme is shown and the numbering system is used in connection with this application. Ye et al., Biochim. The corresponding acid can be determined by reference to Biophys Acta 1339 (1997) 39-52.
[0017]
With respect to possible changes in the amino acid residue corresponding to residue 357 in the Photonus pyralis luciferase, most wild-type sequences have an acid residue (aspartic acid or glutamic acid) at this position. An exception to this is a form of luciferase of Photouris pennsylvanica, in which the corresponding residue (355) is a nonpolar residue valine, or in a form of luciferase of Phrixothrix, the corresponding position is Pv_GROr Ph_REV354 in the middle, and L354 leucine. Thus, in general, the amino acids used as substitution amino acids at this position are other than aspartic acid, glutamic acid, valine or leucine.
[0018]
Thus, in many cases acidic amino acid residues are charged with basic amino acids such as lysine or arginine, nonpolar amino acids such as leucine, valine or isoleucine, tyrosine, asparagine, glutamine, phenylalanine, serine, tryptophan or threonine. Replaced with non-acidic residues including no polar amino acids. In particular, this can be replaced by an uncharged polar amino acid such as tyrosine, asparagine, serine or threonine. Particularly preferred amino acid residues for substitution at this position are tyrosine, phenylalanine or tryptophan, with tyrosine being most preferred. Generally speaking, the aromatic residue at this position causes a maximum shift and can also maintain thermal stability.
[0019]
If the wild type sequence contains non-acidic amino acid residues at this position, they are appropriately mutated to different non-acidic residues.
[0020]
By mutating the enzyme in this way, it has been found that the wavelength of light emitted by luciferase shifts to 50 nm towards the red end of the spectrum in some cases. Thus, the D357Y mutant Photinus pyralis luciferase emits light at a wavelength of about 612 nm compared to the wild-type enzyme that emits light at a wavelength of 562 nm.
[0021]
A wavelength shift of 50 nm has considerable capacity for use in assay applications since this intensity shift can be easily determined spectrally. For example, as described in WO95 / 18853, different color luciferases can be used as reporter molecules in gene expression studies, which allows two or more genes to be monitored simultaneously. A number of analyte tests can also be performed using luciferase as a label.
[0022]
The fact that the light in this case is deep red is particularly useful in assay methods. When analyzing solutions for ATP containing dyes or other compounds that can absorb short wavelengths of light, the red mutant would be useful. For example, a red solution will not absorb red light. Examples of red solutions that are often subject to such analysis include solutions of blood samples or eukaryotic cell culture media that may contain a red pH indicator.
[0023]
When using a mixture of colorimetric agents such as luciferase, the ability to produce a deep red signal may be useful, especially when other reagents in the sample produce a green signal. A photomultiplier tube used in photoelectrode spectral analysis can be set to detect one or two peaks produced in one sample. In other words, it is possible to distinguish the photon flow from the red and the green emitter in the same sample.
[0024]
Furthermore, it has been found that the shift in wavelength can be affected by the presence of cofactor coenzyme A (CoA). This feature creates the possibility of using this enzyme in assays for cofactors.
[0025]
The effect of cofactor coenzyme A on the in vitro spectrum of emitted light was examined as described below. As the coenzyme A concentration increases, the spectrum distribution changes, and at the maximum CoA concentration, the wavelength in the region from 590 to 630 nm with a distinct peak at 610 nm becomes the main spectrum.
[0026]
Thus, according to another aspect of the invention, an assay is provided for determining the presence of CoA in a sample. This assay involves adding other reagents needed to trigger the luciferase / luciferin reaction with the aforementioned luciferase to a sample suspected of containing CoA, measuring the wavelength of light emitted from the sample, and Associated with the presence or absence of CoA.
[0027]
Such assays can be useful in detecting the growth or activity status of cells, eg, microorganisms or eukaryotic cells.
[0028]
For example, the concentration of CoA in E. coli cells is relatively high and varies significantly with metabolic conditions. Since the wavelength of luminescence changes depending on the concentration of CoA, the mutant enzyme of the present invention can be used to monitor the metabolic status of an organism, particularly the concentration of CoA in vivo. Such an assay may be particularly useful in situations where CoA is an important major metabolite in the production of antibiotics (eg, Streptomyces). Cellular CoA concentration is also an important indicator of fatty acid biosynthesis and varies with cell starvation conditions. Some metabolic disorders such as carcinogenesis and diabetes exhibit abnormalities in fatty acid metabolites and thus abnormal CoA levels. The assay of the present invention can be used in the diagnosis of such conditions. For example, by measuring the wavelength of light emitted from the luciferase of the invention used in the assay, cell samples such as blood samples from a patient and CoA levels can be measured. This result can be compared to the result obtained from a sample of healthy cells to determine if the wavelength changes and there is an altered CoA level. This can be an indication of the patient's disease state. Prior to the assay, the cells are appropriately lysed using known lysing agents.
[0029]
The amino acid residue at position 357 is believed to be critically associated with the binding site of coenzyme A. The surface of the luciferase enzyme was outlined to 1 angstrom resolution (using Tripos Ltd.'s SYBL protein nodding software) and a small polar pocket was found. This pocket appears to be lined by residues H310, E354 and D357 and is measured to be 8-10 Å. Viewed from the top of the molecule, this pocket appears to be part of a larger pocket lined by residues H310, E354, D357 and I232. Residues H310 and E354 appear to form a bridge across the cleft, giving the appearance of two small pockets (see FIG. 8).
[0030]
Without being bound by theory, when the enzyme is in solution, the bridge residue is flexible enough to break off and allow a larger pocket (up to 12 mm deep and 8 mm wide) to allow CoA binding. It seems to provide. This is consistent with the energy calculation.
[0031]
When E. coli cells expressing the firefly luciferase variants of the present invention were grown on different carbon sources, a change in the spectrum of light emitted in vivo was observed. Conversion from enriched medium (LB) to limited minimal medium with acetate or glucose as the only carbon source shifted the emitted light to longer wavelengths and reduced the contribution from shorter wavelengths. This would provide another means for assay purposes to adjust the wavelength of emitted light.
[0032]
A mutation at position 357 in this protein was found to result in improved thermal stability.
[0033]
The protein may contain other mutations in the sequence, provided that the luciferase activity of the protein is not significantly impaired. This mutation appropriately improves the properties of the enzyme or, to some extent, better adapts the enzyme to the desired purpose. This means that the mutation is thermostable and / or color-shifting in nature and / or K for this enzyme's ATP._mLet's mean that will improve. Examples of mutations that cause a color shift are described in WO95 / 18853. K_mMutations affecting the value are described, for example, in WO 96/22376 and International Patent Application No. PCT / GB98 / 01026.
[0034]
In general, mutation effects have been found to be additive in terms of changes in properties.
[0035]
The mutant luciferase of the present invention may contain other specific mutations that increase thermal stability compared to wild-type luciferase. In detail,
(A) the amino acid residue corresponding to amino acid 354 of Photinus pyralis luciferase (356 in Luciola luciferase) is mutated;
(B) the amino acid residue corresponding to position 215 in Photinus pyralis luciferase or (217 in Luciola luciferase) is a different hydrophobic amino acid;
(C) an amino acid residue corresponding to residue 214 in Photinus pyralis luciferase or residue 216 of Luciola mingrelica, Luciola cruciata or Luciola lateralis luciferase;
(D) residue 232 in Photinus pyralis luciferase or an amino acid residue corresponding to residue 234 of Luciola mingleica, Luciola cruciata or Luciola lateralis luciferase;
(E) an amino acid residue corresponding to residue 295 in Photinus pyralis luciferase or residue 297 of Luciola mingrelica, Luciola cruciata or Luciola lateralis luciferase;
(F) the amino acid residue corresponding to amino acid 14 in Photinus pyralis luciferase, or residue 16 of Lucio minarelica, or 17 of Luciola cruciata or Luciola lateralis;
(G) the amino acid residue in Photinus pyralis luciferase, or the amino acid residue corresponding to residue 37 of Luciola mingrelica, or residue 38 of Luciola cruciata or Luciola lateralis;
(H) an amino acid residue corresponding to amino acid residue 105 of Photinus pyralis luciferase, or residue 106 of Luciola mingrelica, 107 of Luciola cruciata, or 108 of the Luciola lateralis gene;
(I) amino acid residue 234 in Photinus pyralis luciferase or amino acid residue corresponding to residue 236 of Lucio minarelica, Lucio cruciata or Lucio la lateralis;
(J) the amino acid residue 420 in Photinus pyralis luciferase or the amino acid residue corresponding to residue 422 of Lucio minarelica, Lucio cruciata or Lucio la lateralis;
(K) amino acid residue 310 in Photinus pyralis luciferase or an amino acid residue corresponding to residue 312 of Luciola mingrelica, Luciola cruciata or Luciola lateralis;
Unlike amino acids in which at least one of the corresponding wild-type sequences is present, this luciferase enzyme has increased thermal stability compared to an enzyme having the corresponding amino acid at this position of the wild-type luciferase.
[0036]
Thus, preferred examples of proteins of the present invention include two or more amino acids, such as up to 100 amino acid residues, preferably no more than 40 amino acids, more preferably up to 30 amino acids, A mutated wild type luciferase that differs from the amino acid at the corresponding position in the appropriate wild type enzyme.
[0037]
Thus, in a preferred embodiment, the protein of the invention comprises a Photinus pyralis luciferase and, in addition to the mutation at position 357 described above, comprises at least one of the following:
[0038]
(A) the amino acid residue corresponding to amino acid 354 of Photinus pyralis luciferase is other than glutamate,
(B) the amino acid residue corresponding to position 215 in Photinus pyralis luciferase is a hydrophobic amino acid other than alanine,
(C) the amino acid residue corresponding to residue 214 in Photinus pyralis luciferase is other than threonine;
(D) the amino acid residue corresponding to residue 232 in Photinus pyralis luciferase is other than isoleucine,
(E) the amino acid residue corresponding to residue 295 in Photinus pyralis luciferase is other than phenylalanine;
(F) the amino acid residue corresponding to amino acid 14 of Photinus pyralis luciferase is other than phenylalanine;
(G) the amino acid residue corresponding to amino acid 35 of Photinus pyralis luciferase is other than leucine;
(H) the amino acid residue corresponding to amino acid residue 105 of Photinus pyralis luciferase is other than alanine,
(I) the amino acid residue corresponding to amino acid residue 234 of Photinus pyralis luciferase is other than aspartic acid;
(J) the amino acid residue corresponding to amino acid residue 420 of Photinus pyralis luciferase is other than serine,
(K) The amino acid residue corresponding to amino acid residue 310 of Photinus pyralis luciferase is other than histidine.
[0039]
Alternatively, the protein of the present invention includes a luciferase sequence of a protein of Lucio mingrelica, Luciola cruciata or Luciola lateralis enzyme, and includes at least one of the following in addition to the aforementioned mutation at position 359.
[0040]
(A) the amino acid residue corresponding to amino acid 356 of Photinus pyralis luciferase is other than glutamate,
(B) the amino acid residue corresponding to position 215 in Photinus pyralis luciferase is a hydrophobic amino acid other than alanine or threonine;
(C) the amino acid residue corresponding to residue 216 in the luciferase of Luciola mingrelica, Luciola cruciata or Luciola lateralis is glycine (if the sequence is based on Luciola mingrelica) or paraffin (if the sequence is Luciolaculac) Is,
(D) the amino acid residue corresponding to residue 234 in Luciola mingrelica, Luciola cruciata or Luciola lateralis luciferase is other than serine,
(E) the amino acid residue corresponding to residue 297 in Luciola mingrelica, Luciola cruciata or Luciola lateralis luciferase is other than leucine,
(F) the amino acid residue corresponding to amino acid 16 of Luciola mingrelica, or amino acid 17 of Luciola cruciata or Luciola lateralis is other than phenylalanine;
(G) the amino acid residue corresponding to residue 37 of Luciola mingrelica, or 38 of Luciola cruciata or Luciola lateralis is other than lysine;
(H) the amino acid residue 106 corresponding to the amino acid residue 106 of Luciola mingrelica, the amino acid residue 107 of Luciola cruciata, or the amino acid residue 108 of Luciola lateralis is other than glycine;
(I) the amino acid residue corresponding to amino acid residue 236 of Lucio minarelica, Luciola cruciata or Luciola lateralis is other than glycine,
(J) the amino acid residue corresponding to residue 422 of Lucio minarelica, Luciola cruciata or Luciola lateralis is other than threonine,
(K) the amino acid residue corresponding to the amino acid residue 312 of Luciola mingrelica, Luciola cruciata or Luciola lateralis is threonine (in the case of a Luciola mingrelica-based sequence) or valine (Luciola cruciata or Luca
[0041]
In either case, the specific substituted amino acid that improves the thermal stability can be determined by the general method exemplified below. In each case, different substitutions could improve thermal stability. As will be appreciated by those skilled in the art, substitutions can be made by site-directed mutation of DNA encoding the original protein or an appropriately mutated protein. In this case, the present invention involves specifying a position with thermal stability.
[0042]
In general, however, it may be desirable to consider substituting an amino acid of a different nature than the wild type amino acid. Thus, it may be preferable to be able to substitute hydrophilic amino acid residues with hydrophobic amino acid residues (and vice versa). Similarly, acidic amino acid residues can be substituted with basic residues.
[0043]
For example, the protein may comprise a protein having luciferase activity and at least 60% similarity to a luciferase from Photinus pyralis, Lucio minarelica, Luciola cruciata or Luciola lateralis enzyme. In the sequence of this enzyme, at least one of the following is mutated,
(A) the amino acid residue corresponding to residue 214 of Photinus pyralis luciferase 214, Lucio mininglica, Luciola cruciata or Luciola lateralis luciferase is mutated, and a luciferase of Photinus pyralis
(B) an amino acid residue corresponding to residue 232 of Photinus pyralis luciferase and amino acid residue corresponding to residue 234 of Lucio mininglica, Luciola cruciata or Luciola lateralis luciferase is an isoleu of leufer,
(C) the amino acid residue corresponding to residue 295 of Photinus pyralis luciferase and the residue 297 of Lucio mininglica, Luciola cruciata or Luciola lateralis luciferase, for example, Phenylus pyralis in the case of Photinus pyralilase. This luciferase enzyme has increased thermal stability compared to wild-type luciferase.
[0044]
All sequences of the various luciferases indicate that they have a high similarity between them and are highly conserved. This means that the corresponding region between the sequences can be easily determined by examining the sequence and the most similar regions can be detected. However, if necessary, using commercially available software (see, eg, “Bestfit” from the University of Wisconsin Genetics Computer Group; see Devueux et al. (1984) Nucleic Acid Research 12: 387-395). Corresponding regions or specific amino acids can be determined. Alternatively or additionally, L. Ye et al., Biochim. The corresponding acid can be determined by reference to Biophys Acta 1339 (1997) 39-52.
[0045]
For possible changes of the amino acid residue corresponding to residue 214 in Photinus pyralis luciferase, the polar amino acid threonine is a non-polar amino acid such as alanine, glycine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan or cysteine Is replaced appropriately. A particularly preferred substitution for the threonine residue, corresponding to residue 214 in Photinus pyralis, is alanine. A more preferred substitution is cysteine. However, different polar residues such as asparagine at this position may improve the thermal stability of the corresponding enzyme with threonine at this position. Wild-type luciferase glycine to other amino acids appearing at this position in the enzyme (Luciola mingrelica, Hotaria parvula), asparagine (Pyrophorus plagiophthalamus, GR, YG, YE and OR, Luciola cruciata, Luciola lateralis, Lampyris noctiluca, Pyrocelia miyako Photuris pennsylvanica LY, KW, J19) and serine (Prixothix). Advantageously, these can be substituted with nonpolar or different nonpolar side chains such as alanine and cysteine.
[0046]
With respect to possible changes in the amino acid residue corresponding to residue 232 in Photinus pyralis luciferase, the nonpolar amino acid isoleucine is a different nonpolar such as alanine, glycine, valine, leucine, proline, phenylalanine, methionine, tryptophan or cysteine. Replaced appropriately with amino acids. Other amino acids that appear at this position in the wild type luciferase enzyme include serine and asparagine. Suitably, these polar residues are replaced with nonpolar residues as described above. A particularly preferred substitution for residue 232 in the Photinus pyralis luciferase and the residue corresponding to residue 234 of the Lucio mininglica, Luciola cruciata or Luciola lateralis group is alanine.
[0047]
Changes in amino acid residues corresponding to residue 295 in Photinus pyralis luciferase, and residue 297 in Lucio mininglica, Luciola luciata or Luciola lateralis luciferase affect the thermal stability of the protein, where x Corresponding to position 292 in the luciferase of). Generally, the amino acid at this position is the nonpolar amino acid phenylalanine or leucine. These are appropriately changed to different nonpolar amino acids. For example, in Photinus pyralis, the nonpolar amino acid phenylalanine is suitably replaced with a different nonpolar amino acid such as alanine, leucine, glycine, valine, isoleucine, proline, methionine, tryptophan or cysteine. A particularly preferred substitution for the phenylalanine residue corresponding to residue 214 in Photinus pyralis luciferase is leucine.
[0048]
Variations in amino acid residues corresponding to amino acid 14 of Photinus pyralis luciferase, or amino acid 16 or 17 of Luciola luciferase (13 of Phrixothrix luciferase) are also possible. This amino acid residue (usually phenylalanine but may be leucine, serine, arginine or in some cases tyrosine) is a different amino acid, especially alanine, valine, leucine, isoleucine, proline, methionine or tryptophan, preferably alanine Change appropriately to different non-polar amino acids such as.
[0049]
Mutations in amino acid residues corresponding to amino acid 35 of Photinus pyralis luciferase, or amino acid residue 37 of Lucio miningella luciferase (38 in other Luciola spp.) Would also be effective. This amino acid varies between wild-type enzymes and may contain lysine (Phototinus pyralis) as well as lysine, histidine, glycine, alanine, glutamine and aspartic acid at this position. Suitably the amino residue at this position is substituted with a non-polar amino acid residue such as alanine, valine, phenylalanine, isoleucine, proline, methionine or tryptophan or a different non-polar amino acid. The preferred amino acid at this position is alanine, which differs from the wild type enzyme.
[0050]
It is preferred that the mutation in the amino acid corresponding to position 14 of the sequence of Photinus pyralis and / or the mutation in the amino acid residue corresponding to amino acid 35 of Photinus pyralis luciferase is not the only mutation in the enzyme.
[0051]
These are suitably accompanied by other mutations as described above, in particular mutations at positions corresponding to positions 214, 395 or 232 of Photinus pyralis luciferase.
[0052]
Corresponding to residue 105 in Photinus pyralis luciferase, and residue 106 in Luciola mingrelica, or residue 107 in Luciola cruciata, or residue 108 in Luciola lateralis luciferase (amino acid 102 in Phrixothix) Will affect the thermal stability of the protein. Generally, the amino acid at this position is the nonpolar amino acid alanine or glycine, or serine in Phrixothix. These are appropriately changed to different non-polar amino acids. For example, in Photinus pyralis, the nonpolar amino acid alanine is appropriately replaced with a different nonpolar amino acid such as phenylalanine, leucine, glycine, valine, isoleucine, proline, methionine, tryptophan. A particularly preferred substitution for the alanine residue corresponding to residue 105 in Photinus pyralis luciferase is valine.
[0053]
Changes in the heat of the amino acid residue corresponding to residue 234 in Photinus pyralis luciferase and residue 236 in Lucifera mingrelica, Luciola cruciata or Luciola lateralis luciferase (231 in Phrixothix), Let's reach. In general, the amino acid at this position is aspartic acid or glycine, and may be glutamine or threonine. These are appropriately changed to non-polar or appropriate different non-polar amino acids. For example, in Photinus pyralis, the amino acid residue is aspartic acid and is appropriately replaced with a non-polar amino acid such as alanine, leucine, glycine, valine, isoleucine, proline, methionine or tryptophan. A particularly preferred substitution for the phenylalanine residue corresponding to residue 234 in Photinus pyralis luciferase is glycine. If a nonpolar amino acid residue, such as glycine, is present at this position (eg in Luciola luciferase), it could be replaced with a different nonpolar amino acid.
[0054]
Residue 420 in Photinus pyralis luciferase, and residue 422 in Lucio mininglica, Luciola cruciata or Luciola lateralis luciferase (residue 417 in Phrixothix green and the red in protein corresponding to Phriox4) It will affect the thermal stability of. In general, the amino acid at this position is the uncharged polar amino acid serine or threonine or glycine. These are appropriately changed to different uncharged polar amino acids. For example, in Photinus pyralis, serine could be appropriately replaced with asparagine, glutamine, threonine or tyrosine, especially threonine.
[0055]
Changes in amino acid residues corresponding to residue 310 in Photinus pyralis luciferase, and residue 312 in Luciola mingrelica, Luciola cruciata, or Luciola lateralis luciferase will affect the thermal stability of the protein. The amino acid residue at this position varies between known luciferase proteins, including Photinus pyralis, Pyroceria miyako, Lampyris noctiluca, and in some forms of Photuris penicilliva luciferase, histidine, It is threonine for luciferase (which is amino acid 307), valine for Luciola cruciata and Luciola lateralis, and asparagine for certain Pyrophorus plagiophthalamus luciferases. Thus, in general, the amino acid at this position is a hydrophilic amino acid that can be changed to a different amino acid residue that increases the thermal stability of the enzyme. A particularly preferred substitution for the histidine residue corresponding to residue 310 in Photinus pyralis luciferase is arginine.
[0056]
Other mutations may be present in this enzyme. For example, in a preferred embodiment, the protein has an amino acid residue at this position corresponding to amino acid 354 of Photinus pyralis luciferase (356 in Luciola luciferase), altered from glutamate, in particular glycine, proline or aspartic acid. It also has an amino acid at this position changed to an amino acid other than. Suitably the amino acid at this position is tryptophan, valine, leucine, isoleucine and asparagine, most preferably lysine or arginine. This mutation is described in WO95 / 25798. A hydrophobic residue at this position has been found to improve the wavelength shift of the enzyme, and is further highly hydrophobic (V or I), polar (N) or positively charged at position 354 (K Or R) The presence of amino acids improves thermal stability.
[0057]
In other embodiments, as described in EP-A-052448, the protein is a hydrophobic amino acid, in particular isoleucine, in a position corresponding to amino acid 217 in Luciola luciferase (215 in Phototinus pyralis). Has amino acids changed to leucine or valine.
[0058]
The proteins of the present invention include both wild type and recombinant luciferase enzymes. These in the sense that at least 60% of the amino acids present in the wild-type enzyme are present in the protein of the present invention, and at least a wild-type sequence such as the sequence of Photinus pyralis, Lucio minarelica, Luciola cruciata or Luciola lateralis enzyme. 60% similarity. Such proteins may have a greater similarity to the aforementioned wild type enzyme, in particular at least 70%, more preferably at least 80% and most preferably at least 90%. Similar proteins are of this type and include allelic variants, proteins from other insect species, and recombinantly produced enzymes. These proteins can be easily identified in that they are encoded by a nucleic acid that hybridizes under stringent hybridization conditions with a sequence encoding a wild type enzyme. Such conditions will be well understood by those skilled in the art. Examples are given in Sambrook et al. (1989) Molecular Cloning, Cold Spring Harbor Laboratory Press. In general, low stringency conditions are defined as 3 × SCC from about ambient to about 65 ° C., and high stringency conditions are defined as 0.1 × SCC at about 65 ° C. SSC is the name of a buffer of 0.15M NaCl, 0.015M trisodium citrate. 3xSCC is three times stronger than SSC.
[0059]
Specifically, the multiple alignment method described by Lipman and Pearson (using Lipman, DJ and Pearson, WR (1985) Rapid and Sensitive Protein Similarity Science, Science, vol 227, pp 1435-1441). Thus, the similarity between a specific sequence and the sequence of the present invention can be evaluated. An “optimized” percentage score should be calculated using the following parameters for the Lipman-Pearson algorithm. ktup = 1, gap penalty = 4 and gap penalty length = 12. The sequence for assessing similarity should be used as the “test sequence”, which is the base sequence for comparison, such as the sequence of Photinus pyralis or any other sequence recorded in Ye et al. Means that this should be included in this algorithm.
[0060]
A specific example of a protein of the invention is a wild type luciferase sequence having one or more mutations as described above.
[0061]
Furthermore, the present invention provides a nucleic acid encoding the aforementioned luciferase. Suitably the nucleic acid is based on a wild type sequence well known in the art. Suitable mutations for making the desired mutation in the amino acid sequence will be readily apparent based on knowledge of the genetic code.
[0062]
In a preferred embodiment of the invention, the nucleic acid is a synthetic gene. Suitably, a synthetic gene is engineered to remove codons that are rarely found in highly expressed genes from common expression hosts such as E. coli, and at the same time in genes encoding beetle luciferases. Avoid introducing codons that are rarely seen. This approach ensures that this new gene has optimal codon usage for both E. coli and insect expression systems.
[0063]
For example, whenever possible, the codons for the amino acids arg, leu, ile, gly and pro are represented as CGT or CGC (arg), CTG, CTT or CTC (leu), ATC or ATT (ile), GGT or GGC (gly), And CCG, CCA or CCT (pro), thus removing rare codons. In the case of the synthetic gene exemplified below (SEQ ID NO: 1) and FIG. 14, this resulted in a total of 139 silent mutations that produced 62 new general codons (11% of the total). The first 8 nucleotides shown in FIG. 14 form part of the ribosome binding site and are therefore not encoded. The coding sequence begins with a methionine residue indicated by an upward arrow. This coding sequence and very similar sequences, for example sequences having at least 90% similarity, preferably 95% similarity, form a preferred embodiment of the present invention.
[0064]
Another useful property that can be used when generating a synthetic assembly is the introduction of new unique restriction sites. These sites make gene mutagenesis, in particular combinatorial alkaset mutations easier and more efficient. Specifically, it may be desirable to create a unique restriction site in the cDNA encoding subdomain B in the enzyme. In addition, it may be advantageous to create a unique restriction site at the 3 'end of the gene to allow easy fusion and / or removal of the peroxome target sequence.
[0065]
In the examples that follow, nine new unique restriction sites have been engineered (mostly the third in the middle of the gene), and a unique Hind III site is created at the 3 'end of the gene, allowing simple C-terminal fusions. (FIG. 12).
[0066]
Finally, the use of synthetic genes allows for the introduction of mutations to increase the thermal stability of the gene product or otherwise modify the product properties as desired. For example, in the examples that follow, three mutations that are not silent were engineered to introduce thermostable amino acid changes T214C, E354K, and D357F into the polypeptide.
[0067]
The nucleic acid of the present invention is appropriately introduced into an expression vector such as a plasmid under the control of regulatory elements such as a promoter, enhancer, and terminator. These vectors then allow the cells to express the desired luciferase enzyme in order to transform host cells, eg prokaryotic or eukaryotic cells such as plant or animal cells, in particular prokaryotic cells such as E. coli. Can be used for The luciferase enzyme will be produced by culturing the cells thus transformed using conditions well known in the art. This can then be separated from the culture medium. When the cells are plant cells or animal cells, plants or animals can be propagated from the cells. Therefore, protein can be extracted from plants, and in the case of transgenic animals, protein can be recovered from milk. Vectors, transformed cells, transgenic plants and animals, and methods of producing enzymes by culturing these cells all form other aspects of the invention.
[0068]
The Photinus pyralis D357Y mutant luciferase was generated by random mutagenesis as described below. A mutation at one point in D357Y produced a large color shift in the wavelength of the emitted light and was found to be more thermostable than wild type luciferase. Further investigation reveals that a range of substitutions at this position produces excellent thermal stability and / or large color shifts.
[0069]
Specific examples of mutants of Photinus pyralis that fall within the scope of the present invention include the following, or any equivalent of these other luciferases.
[0070]
[Table 1]

[0071]
The mutation for preparing the mutant was introduced into the luciferase gene on the plasmid pET23 by site-directed mutagenesis, (PCR) or combinatorial alkaset mutation. The oligonucleotides added to the PCR reaction to make the appropriate mutations are shown below.
[0072]
It has been previously reported that the effects of point mutations at positions 354 and 215 are additive. The present invention provides the possibility of combining three or more such mutations to provide high thermal stability in mutant enzymes with large color shifts.
[0073]
The luciferase proteins of the present invention may be advantageously used in any bioluminescence assay that uses the luciferase / luciferin reaction as a signal means. There are many such assays known in the literature. Thus, this protein will be included in kits made for the purpose of performing such assays. Optionally, luciferin and any other reagents required to perform a particular assay are also included.
[0074]
The present invention will now be described in detail by way of example with reference to the accompanying drawings.
[0075]
FIG. 1 is a logarithmic graph showing% residual activity versus time of incubation of several mutant enzymes according to the invention at 45 ° C.
[0076]
FIG. 2 shows the spectral peaks obtained by incubating E. coli cells expressing the luciferase enzyme in citrate buffer with D-luciferin. In this case, the enzymes used were (a) recombinant wild-type Photinus pyralis luciferase, (b) D357K mutant, (c) D357N mutant, (d) D357W mutant, (e) D357I mutant, (f) D357F mutant, (g) D357Y mutant, and (h) double mutant E354I + D357Y.
[0077]
FIG. 3 is a graph showing% residual activity vs. time for three mutant enzymes E354I, D357Y and double mutant (DM) E354I / D357Y.
[0078]
FIG. 4 shows the emission spectra of (a) recombinant wild-type enzyme and (b) double mutant (DM) E354I / D357Y.
[0079]
FIG. 5 is a graph showing the rate decay of photon luminescence of recombinant wild type (♦) r-wt and D357K mutant enzyme (■).
[0080]
FIG. 6 shows a molecular model diagram showing possible CoA binding pockets in the luciferase enzyme.
[0081]
FIG. 7 shows mutant P. a. Grown on (a) LB, (b) grown on minimal medium and sodium acetate, (c) grown on minimal medium and glucose. Figure 2 shows in vivo bioluminescence spectra emitted by E. coli cells expressing pyralis luciferase D357Y.
[0082]
FIG. 8 shows mutant P. (a) grown on LB, (b) grown on minimal medium and sodium acetate, (c) grown on minimal medium and glucose. Figure 2 shows in vivo bioluminescence spectra emitted by E. coli cells expressing Pyralis luciferase E354K / D357M.
[0083]
FIG. FIG. 6 is a graph showing the effect of CoA on the spectral distribution of light emitted by pyralis luciferase D357Y.
[0084]
FIG. FIG. 6 is a graph showing normalized data of the effect of CoA on the spectral distribution of light emitted by pyralis luciferase D357Y.
[0085]
FIG. 12 is a graph showing the effect of CoA (FIG. 11a) and normalized data (FIG. 11b) on the spectral distribution of light emitted by pyralis luciferase E354I / D357Y.
[0086]
FIG. 12 shows the restriction site modifications used in the construction of the synthetic luciferase gene.
[0087]
FIG. 13 shows the construct used in the synthesis of the luciferase gene.
[0088]
FIG. 14 begins with the synthetic luciferase gene cDNA sequence (SEQ ID NO: 1), which forms part of the ribosome binding site and includes non-coding nucleotides 1-8, and a methionine residue indicated by an upward arrow. Shows the encoded amino acid sequence (SEQ ID NO: 2).
[0089]
FIG. 15 shows the thermal stability at 50 ° C. of mutants, including mutants encoded by synthetic genes.
[0090]
Example 1
Identification and characterization of mutant luciferases
Error-pron PCR [M. Fromt et al., Anal. Biochem. (1995) 224, 347-353], two libraries of firefly (Photinus pyralis) luciferase were prepared. One library consisting of error-pron PCR products of the full-length luc gene was cloned into the T7 expression system pET23a (Novagen Inc., Madison, WI, USA). A second library of error-pron PCR products of a short fragment of the luc gene covering 199-35 amino acids was cloned into the vector pBSK (+) (Stratagene, La Jolla, CA, US). A).
[0091]
The pET23a library was expressed in E. coli strain BL21 (DE3) (E. coli B F dcm opt hsdS (r_B ⁻m_B ⁻) Galλ (DE3)).
[0092]
The pBSK (+) library was transferred to HB101 E. coli cells (supE44 aral4 galK2 lacY1 Δ (gpt-proA) 62 rpsL20 (Str^r) Xyl-5 mtl-1 recAl3 Δ (mrcC-mrr) HsdS⁻(R⁻m⁻)). Both pET23a and pBSK (+) carry the gene for β-lactamase and confer ampicillin resistance to E. coli cells carrying this plasmid.
[0093]
BIORAD E.I. The generated library was transformed with the generated library by electroporation using E. coli Pulser and grown overnight at 37 ° C. on LB agar containing ampicillin at a concentration of 50 μg / ml. Cells were transferred to nylon membranes (Osmonics, Minnetonka, Minnesota, USA) and sprayed with luciferin solution (500 μM D-luciferin, potassium salt in sodium citrate buffer 100 mM, pH 5.0). Colonies were observed using AlphaImager ™ 1200 Document and Analysis System (Flowgen, Richfield, Staffire, UK). This captures the bioluminescence emitted at a particular time, producing an image of the light emitted by the colony. Luminescence brightness was recorded as an indicator of the thermostability of luciferase.
[0094]
The colonies were then selected for thermal stability. Colonies were selected based on the brightness of the emitted light and isolated for further characterization. In some screens, E. coli colonies were incubated at 42 ° C. for 2 hours prior to selection, so that thermostable mutants could be selected. Colonies isolated from the initial selection were mounted on nylon membranes and grown overnight on LB medium containing ampicillin. Patches were sprayed with luciferin solution and observed in AlphaImager ™. This second selection helped positively identify clones for in vitro analysis of luciferase activity. E. coli clones expressing potential thermostable enzymes were assayed in vitro for luciferase activity and thermostability.
[0095]
In vitro assays for luciferase activity were performed at room temperature using the Promega Luciferase Assay System (Promega Corporation, Madison, Wis., USA).
[0096]
The luciferase reaction was initiated by adding 10 μl of crude cell extract to Promega Luciferase Assay Cocktail (diluted 1: 2). The resulting bioluminescence was measured using a Biotrace M3 luminometer.
[0097]
Promega technical bulletin no. Crude cell extracts were prepared as described in 101. Cell culture lysis reagent (25 mM trisphosphate pH 7.8, 2 mM dithiothreitol (DTT), 2 mM 1,2-diaminocyclohexane-N, N, N ′, N′-tetraacetic acid, 10% glycerol, 1% Triton X-100, 1.25 mg / ml hen lysozyme) and aliquots of E. coli cultured overnight at room temperature for 10 minutes. The crude lysate was then stored on ice prior to assay.
[0098]
The properties of the enzyme were further tested in a time-dependent inactivation experiment. Eppendorf tubes containing 50 μl aliquots of crude cell extract were incubated at a given temperature in a water bath. At several time points set, the tubes were removed and cooled on ice before the assay. Residual luciferase activity was expressed as a percentage of the original activity.
[0099]
Plot a logarithmic graph of% residual activity versus incubation time, t_1/2Used to calculate the value. T_1/2Is the time it takes for an enzyme to lose 50% of its original activity after incubation at a given temperature. From the logarithmic graph of time against% residual activity, T in the crude extract at 37 ° C._1/2Values (time for activity to fall to 50% of original activity) were determined (not shown).
[0100]
Plasmid DNA from an E. coli clone expressing the most thermostable luciferase determined as described above was sequenced to determine the mutation responsible for the enzyme thermostability.
[0101]
Plasmid DNA was made using the QIAGEN QIAprep Spin Miniprep Kit (QIAGEN Ltd, Crawley, W. Sussex, UK) followed by a protocol for using microcentrifugation (QIAprep Miniprep Handbook 04/98).
[0102]
All DNA sequencing was performed using the ABI PRISM ™ 377 DNA Sequencer and the dideoxy chain termination method [F. Sanger et al., Proc. Natl. Acad. Sci. U. S. Brach using an ABI PRISM ™ BigDye ™ Terminator Cycler Sequence Ready Reaction Kit (Perkin Elmer Applied Biosystems) based on A 74, (1977) 5463-5467].^nix, Cambridge, UK.
[0103]
As a result of this work, a novel mutant D357Y was identified.
[0104]
Crystal structure of luciferase [E. Conti et al., Structure 4 (1996) 287-298] that position 357 is close to the surface of the protein and is close to position 354, which may affect both thermal stability and spectral properties. Indicates. This indicates that this region may be important in terms of enzyme thermal stability.
[0105]
D357Y is a very thermostable variant, the most thermostable luciferase, with only one amino acid change.
[0106]
Example 2
Site-specific mutations to generate other 357 mutants
To evaluate the different mutants at position 357, site-specific mutations were performed using the Stratagene QuikChange ™ Site-Directed Mutagenesis Kit (Stratagene, La Jolla, Calif., USA). Plasmid pPW601a J54 (PJW, MoD Report, 3/96) was used in all site-specific mutations. All products of the mutagenesis reaction were purified from E. coli strain XL1-Blue [e14⁻(McrA⁻) Δ (mcrCB-hsdSMR-mrr) 171 endA1 supE44 thi-1 gyrA96 relAl lac recB recJ sbcC umuC :: Tn5 (Kan^r) UvrC [F'proAB lacl^qzΔm15 Tn 10 (Tet^r) Amy Cam^r] Was transformed. Sigma-Genosys Ltd. , Cambridge, UK to synthesize oligonucleotide primers and the intelligent doping system [A. R Arkin et al., Bio-technology, (1992) 10, 297-300, W,. Hung et al., Anal. Biochem. (1992) 10, 454-457], and without using individual primers for each amino acid substitution, this was used to design degenerate oligonucleotide primers to identify potential variants. Groups were created.
[0107]
In this way, a library of amino acid substitution luciferase variants was created.
[0108]
The following oligonucleotides (and their complementary partners) were used.
[0109]
[Chemical 1]

[0110]
The library of mutants was screened for thermal stability as described above. The following equation [S. Crimie et al. Biol. Chem. 265 (1990) 18776-187779] was used to calculate the number of colonies picked.
[0111]
N = [1n (1-p)] / [1n ((n-1) / n]
Where N is the number of colonies to be selected, n is the number of possible codons at the target location and P is the probability that each codon in the mixture will be sampled for at least one selection. This calculation was based on P = 0.95. Variants obtained from site-specific mutations were assayed for luciferase activity and characterized in time-dependent heat inactivation experiments.
[0112]
Mutants that are desirable in this way include ampicillin and A₂₆₀It was grown in 400 ml LB medium at 0.5. Luciferase expression was then induced by adding isopropyl β-thiogalactosidase (IPTG) to a final concentration of 1 mM. Cells were incubated for 3 hours with agitation at 30 ° C. and then harvested by centrifugation. The resulting cell pellet was purified according to the B-PER ™ protocol for Maxi-Scale Bacterial Protein Extraction, according to B-PER ™ Protein Extraction Reagent, (Pierce Chemical Company, RochTord, U.M. Resuspended in to make a crude extract. Reconstructed Sigma Protease Inhibitor Cocktail, 500 μl (Product No. P8465, Sigma, Saint Louis, Missouri, USA) was added to the B-PER ™ solution to inhibit endogenous protease. The cell lysate was then centrifuged at 30000g for 30 minutes.
[0113]
The supernatant of the crude extract was fractionated using ammonium sulfate. The fraction precipitated at saturation between 30% and 55% contained luciferase activity. This material was resuspended in 0.5 ml Tris HCl pH 8.0, 1 mM DTT and used for thermal inactivation and spectral experiments.
[0114]
Transformations D357L, T, V, W, R, I, S, K, N and F were introduced. These mutants were characterized in an in vitro heat inactivation experiment of the crude extract.
[0115]
The partially purified extract was diluted in a ratio of 1 to 11 in 50 mM potassium phosphate buffer pH 7.8 containing heat inactivated buffer: saturated ammonium sulfate 10%, 1 mM dithiothreitol and 0.2% BSA.
[0116]
A 110 μl aliquot of the protein solution was incubated at 40 ° C. or 45 ° C. for the set time before the assay and cooled on ice. Luciferase activity was then measured as described in Example 1 using Promega Luciferase Assay Reagent (diluted 1: 2).
[0117]
The results are shown in Tables 2 and 3 and FIG. The T1 / 2 value of the crude extract was determined at 40 ° C. (Table 2) and 45 ° C. (Table 3).
[0118]
[Table 2]

[0119]
[Table 3]

[0120]
All substitutions showed improved thermal stability compared to recombinant wild type.
[0121]
Example 3
Change in wavelength of emitted light
It was also observed that the amino acid substitution at position 357 affects the spectrum of light emitted by the enzyme in vitro. An aliquot (250 μl) of the E. coli cell culture described in Example 2 was grown overnight at 37 ° C., spun down in a microcentrifuge, and the supernatant was removed. Cells expressing different mutant luciferases were incubated in citrate buffer (pH 5.0) containing 150 μl of D-luciferin and SPECTRAmax® Microplate Spectrofluorometer, (Molecular Devices Crop. California, USA). Was used to analyze the light emitted from the in vitro reaction. For mutants D357Y, F and I, a large change in spectral peak and wavelength distribution was observed (FIGS. 2 (a)-(g)). These results are summarized in Table 4 below.
[0122]
In addition, the luminescence of the mutants in vivo was assessed visually in the dark. The D357 mutant showed various colors in its emission spectrum. In particular D357Y, F and I showed a significant shift of the emitted light to longer wavelengths.
[0123]
In some cases (eg, D357F), the change in color of the emitted light is λ_maxIt seems to be due to the difference in the contribution of the visible light from different wavelengths to the spectrum as well as due to the shift of.
[0124]
[Table 4]

[0125]
Λ of in vivo luminescence of certain 357 variants_maxFor comparison, recombinant wild-type (r-wt) enzyme was used. D357Y, F and I showed a significant shift in their wavelength maximum.
[0126]
Example 4
Properties of enzymes in the presence or absence of CoA
D357Y was partially purified by ammonium sulfate precipitation as described in Example 1. This partially purified D357Y enzyme (5 μl) was mixed with 150 μl of Promega Luciferase Assay Reagent. Other aliquots are free of CoA (25 mM tristolysin pH 7.8, 5.0 mM MgSO₄, 0.1 mM EDTA, 2 mM DTT, 470 μM D-luciferin, 530 μM ATP) and mixed with equivalent assay buffer. The emission spectra of the two reactants were measured. This is shown in FIGS.
[0127]
These spectra show significant differences in bioluminescence in the presence or absence of CoA, and λ_maxIndicates a significant shift. The effect of CoA on the kinetics of the luciferase reaction can also be seen by the difference in RLU scale (RLU—relative light units).
[0128]
This difference in luminescence creates the possibility of using this enzyme in assays to detect the presence of CoA.
[0129]
Example 5
Production and properties of double mutants
A double mutant of E354I + D357Y was engineered to investigate any cumulative effects on thermal stability and luminescence color using site-specific mutations as described in Example 2.
[0130]
Dilute partially purified double mutant E354I + D357Y at a ratio of 1 to 11 in heat inactivated buffer: 50 mM potassium phosphate buffer pH 7.8 containing 10% saturated ammonium sulfate 10%, 1 mM dithiothreitol and 0.2% BSA did.
[0131]
A 110 μl aliquot of the protein solution was incubated at 40 ° C. or 45 ° C. for the set time before the assay and cooled on ice. Luciferase activity was then measured as described above using Promega Luciferase Assay Reagent (diluted 1: 2).
[0132]
The double mutant showed a significant increase in thermal stability compared to the single mutants E354I and D357Y, respectively (see FIG. 3). Thermal inactivation experiments of the partially purified double mutant confirmed the increased thermal stability of this mutant, which was 7.7 min t after inactivation at 45 ° C._1/2A value was given.
[0133]
Compared to the individual variants of E354I and D357Y, this double variant showed deeper red emission and was shown to be additive in the color of the emission.
[0134]
Using the assay buffer described in Example 3, the emission spectra of the crude extract of recombinant wild type and double mutant E354I + D357Y were also measured.
[0135]
The emission spectrum measured in vivo is 611 nm λ_maxGave. However, this spectrum greatly contributes to the emission from the red region of the wavelength and gives a deeper red appearance when visualized with the naked eye. The emission spectrum of the crude extract showed a clear change in spectral shape and a 44 nm wavelength shift compared to rWT (see FIG. 4).
[0136]
The emission spectrum of the double mutant in vivo shows both sharpening the peak wavelength bandwidth of emission (613 nm) and reducing the contribution of light wavelength in the range of 540-560 nm.
[0137]
The dramatic effect of these mutations demonstrates the importance of this enzyme to the bioluminescent color of this region.
[0138]
Example 6
Improved photon flow
The in vivo bioluminescence of E. coli cells expressing mutant D357K was observed to be very bright compared to other mutants at this position. The flash kinetics of this enzyme was analyzed using a photometer that can measure the rate of photon emission in time. An aliquot of an E. coli cell-free extract containing recombinant wild-type enzyme or mutant D357 was added to a luciferase assay cocktail that did not contain any reagents (eg, coenzyme A) that facilitated glow kinetics. The rate of decay of photon emission at time (15 seconds) was measured for both enzymes and mutant D357K was observed to be significantly slower (FIG. 4). In other words, the mutant enzyme has a reaction kinetic that is inhibited to a lesser extent than the recombinant wild-type enzyme for at least the first 15 seconds of the reaction.
[0139]
Example 7
Combinatorial alkaset mutation at positions E354 and D357
Step 1
Construction of plasmid pPW601aJ54 for cassette mutation
Two new unique restriction sites were introduced into the luc gene in plasmid pPW601a / J54 using two pairs of synthetic oligonucleotides (see below). A total of 6 silent mutations were introduced into the SpeI and KpnI restriction sites in this gene separated by 63 base pairs. The plasmid containing these new sites was called pPW601aJ54SpeI / KpnI. The presence and proximity of these restriction sites makes it possible to explore the effects of random substitutions at amino acid positions E354 and D357 in the main sequence of firefly luciferase using combinatorial alkaset mutations.
[0140]
[Chemical 2]

[0141]
Nucleotides highlighted in bold lines form an endonuclease recognition site, and those in the upper case form the location of the point mutations necessary to create this site.
[0142]
Step 2
Cassette design and library construction
A pair of synthetic oligonucleotides was synthesized. This oligonucleotide was then annealed to create a double stranded cassette that could be directly ligated to the plasmid pPW601aJ54SpeI / KpnI digested with the new restriction sites. This cassette was designed so that all possible combinations of the 20 naturally occurring amino acids could be introduced at positions E354 and D357 in this primary sequence.
[0143]
[Chemical Formula 3]

2 μg of each of the loop library oligonucleotides was mixed in a buffer containing 50 mM Tris-HCl pH 7.4, 25 mM NaCl and heated to 100 ° C. for 3 minutes. The solution was then slowly cooled to <50 ° C. in a heating block to anneal the complementary sequence. The annealed oligonucleotide was then ligated into plasmid pPW601aJ54SpeI / KpnI and digested with SpeI and KpnI. An aliquot of this ligation reaction was then used to transform E. coli HB101 cells using electroporation. After electroporation, the transformed cells were placed on LB agar plates containing 50 μg / ml ampicillin and grown overnight at 37 ° C. The next day, 869 colonies were randomly picked from the plate and used to inoculate 96 square well plates (Beckman) with 1 ml of LB containing ampicillin. The plate was covered and the cells were grown overnight at 37 ° C. with shaking.
[0144]
Step 3
In vivo selection of randomly selected clones
The next morning, 50 μl aliquots of stationary phase overnight cultures were transferred to two clear plastic round bottom 96 well microtiter plates (Dynex). One plate was covered and incubated on a heated block for 8 minutes (block surface temperature 45 ° C.), while the other block was kept at 37 ° C. Then 50 μl of 100 mM sodium citrate buffer (pH 5.0) containing 0.5 mM D-luciferin is added to the wells and then the plate is transferred to a video camera imager system (Alpha Imager) both in vivo. The luciferase activity in the cells from the plate was detected and recorded at room temperature. The light emitted by the heated and conditioned culture was integrated over 1-2 minutes and the image was recorded on thermal paper film.
[0145]
By determining the brightness of the image recorded on the film, 79 cultures that showed the greatest bioluminescence were selected for the second round of sorting. This time the culture was incubated for 16 minutes on the heating block prior to the assay. Of the 55 clones selected from the in vivo thermostability selection, 25 were selected for in vivo spectral analysis. These clones were grown overnight at 37 ° C. in LB, and 200 μl of the overnight culture was centrifuged the next morning, and the E. coli cell pellet was centrifuged in 100 mM sodium citrate buffer (pH 5) containing 0.5 mM D-luciferin. 0.0) Resuspended in 150 μl. The resuspended cells were then placed in a white plastic microtiter plate and the in vivo bioluminescence spectrum emitted by each of the mutant luciferases was analyzed using a Molecular Devices Spectramax, 96-well plate fluorometer. And analyzed. The results are summarized in Table 1 below.
[0146]
Step 4
Mutation identification
Plasmid DNA was prepared from 25 clones selected by in vivo selection and sequenced using gene specific sequencing primers. Mutations that resulted in amino acid changes at positions 354 and 357 in the primary sequence were identified. One mutant resulted in an amino acid substitution at position I351 (Table 5).
[0147]
[Table 5]

[0148]
Several mutant luciferases were selected from thermostable in vivo assays. Most of these luciferases also showed a large change in the in vivo spectrum of emitted light, and many showed a greater contribution from the long wavelength of light (> 580 nm). Some spectra show a very narrow bandwidth around a single peak at 610-614 nm.
[0149]
Replacement of E354 and D357 with hydrophobic and aromatic amino acids, respectively (eg, E354V, D357Y) results in a large change in the spectrum in vivo, showing a narrow bandwidth unit peak around 612 nm.
[0150]
Example 8
In vivo screening for thermal stability
Cell-free extracts of selected clones were prepared by lysis and the thermostability of luciferase from each extract was measured in a heat inactivation experiment. 50 μl of each extract was placed in an Eppendorf tube and incubated in a water bath heated to 45 ° C. for 4, 9 and 16 minutes. Aliquots were taken at appropriate time points and residual luciferase activity was measured. Table 6 shows the% remaining activity versus time for all mutant enzymes and recombinant wild type.
[0151]
These results show that the most thermostable luciferase is an aromatic amino acid (Y, F or W) at position 357 and a large hydrophobic (V or I), polar (N) or positively charged at position 354. Is a luciferase having a (K or R) amino acid.
[0152]
[Table 6]

[0153]
Example 9
Influence of growth conditions on the in vivo spectrum of emitted light.
The effect of different carbon sources on the spectrum of light emitted from E. coli BL21 (DE3) cells expressing mutant luciferase E354K + D357M (7 described above) was examined.
[0154]
50 ml cell cultures were grown on LB medium to mid-log phase and then harvested by centrifugation. The cell pellet is resuspended in 1 ml sterile distilled water, and then a 100 ul aliquot of this suspension is used to add 5 ml fresh LB, M9 minimal medium + 2 mM sodium acetate or M9 minimal medium + 2 mM glucose into a 25 ml sterile tube. Vaccinated. The culture was continued to grow at 37 ° C. with shaking, and after 90 minutes (D357Y) and 120 minutes (enzyme 7), 200 ul aliquots of cells were taken and centrifuged, and 100 mM containing 0.5 mM D-luciferin was added. Resuspended in 150 ul of sodium citrate buffer (pH 5.0). The resuspended cells were then placed in microtiter plates and the in vivo spectrum of bioluminescence emitted by each of the mutant luciferases was analyzed using a Molecular Devices Spectramax, 96 well plate fluorimeter. The results are shown in FIGS.
[0155]
These results show that from enriched medium (LB) (Figures 7a, 8a) to limited minimal medium with either acetate (Figures 7b, 8b) or glucose (Figures 7c, 8c) as the only carbon source. The change indicates that the emitted light shifts to longer wavelengths and reduces the contribution from shorter wavelengths.
[0156]
Example 10
Purification and spectral characterization of recombinant wild-type and mutant luciferases
Recombinant wild type Phototinus pyralis enzyme and mutant luciferases D357Y and E354I + D357Y were purified to homogeneity to analyze the effect of cofactor coenzyme A on the spectrum of the bioluminescent reaction. All three luciferases were purified as fusions to the 143 amino acid carbohydrate binding module (CBM) from the anaerobic fungus Pyromyces equii. This CBM has been shown to selectively bind to acid-swellable cellulose and soluble carbohydrates galactomanan and glucomanan, forming the basis for a simple single-step affinity purification scheme.
[0157]
Luciferase fused to CBM was able to bind to cellulose in the crude cell-free extract, which was washed and then selectively eluted using soluble polysaccharide. The fusion protein thus purified was used in the assay to measure the wavelength of light emitted in reactions containing different amounts of coenzyme A. 100 μl of assay reagent containing different amounts of coenzyme A (25 mM tristolysin pH 7.8, 5.0 mM MgSO₄, 0.1 mM EDTA, 530 μM ATP and 470 μM D-luciferin) was added enzyme (5 μl). FIGS. 9-11 show the effect of increased coenzyme A concentration on the spectrum of light emitted by purified luciferases D357 and E354I + D357Y.
[0158]
In vivo assays of the spectrum of bioluminescence emitted by E. coli cells fused to the C-terminus of the fungal CBM and expressing firefly luciferase do not show any significant difference from the cells expressing the original luciferase. It was. Similarly, the in vitro assay of the bioluminescence spectrum emitted by a commercial product of purified recombinant luciferase (Promega) was identical to the spectrum emitted by the fusion protein.
[0159]
Therefore, the observed difference is associated with the CoA concentration. As the coenzyme A concentration increases, the spectral distribution changes, and at the maximum CoA concentration, the spectrum is predominantly in the 590-630 nm region with a distinct peak at 610 nm. The spectral shift is most pronounced for the double mutant, with a significant narrowing of the bandwidth around the only peak at a wavelength of 610 nm (FIG. 11).
[0160]
Example 12
Production of synthetic Phototinus pyralis luciferase mutated to have 214C / 354K / 357F
A synthetic luc gene was designed and assembled from oligonucleotide pairs using the synthetic strategy described above. The gene sequence was engineered to produce luciferase with amino acids 214C, 354K and 357F.
[0161]
29 pairs of overlapping synthetic oligonucleotides were synthesized by Sigma-Genosys Ltd, purified by PAGE and ligated into three assemblies of approximately 550 bp (IDRIS 1, 2 and 3, FIG. 13). Each assembly was then ligated separately to the vector pBSK (+) and the resulting construct was used to transform E. coli XL1-Blue cells. Plasmid DNA was prepared from the clone containing the assembled insert and sequenced to confirm the suitability of the ORF. The presence of n-1 oligonucleotides in the assembly (byproduct of oligo synthesis) complicates the construction process. One correct assembly of IDRIS2 was identified by DNA sequencing, and PCR was used to correct one assembly of IDRIS3 containing a single base pair deletion at the 5 'end of the construct. The complete ORF assembly was obtained by ligating the plasmid containing IDRIS1 with a mixture of IDRIS2 and 3.
[0162]
The ligated DNA was then used to transform E. coli XL1-Blue cells and clones expressing the active enzyme were selected using in vivo assays. Several clones were selected and sequenced to confirm the presence and fitness of the synthetic luc gene having the sequence shown in FIG. The complete ORF was called IDRIS (FA).
[0163]
A synthetic gene was assembled between the BamH I and Sal I sites in the polylinker in the vector pBSK (+). In this position, the gene is not in frame with the alpha peptide and is at a considerable distance from the lac promoter. However, sufficient luciferase has been produced to allow preliminary characterization of the enzyme. A crude cell-free extract of E. coli XL1-Blue cells expressing IDRIS (FA) was prepared from those cultured overnight using the Promega lysis method.
[0164]
The thermostability of the enzyme in the extract was then tested at 50 ° C. for 20 minutes and compared to the thermostable mutant E354I + D357Y. The triple mutant with optimized new codons was significantly more thermostable than the mutant E354I + D357Y (FIG. 15).
[Brief description of the drawings]
FIG. 1 is a logarithmic graph showing% remaining activity vs. time of incubation of several mutant enzymes according to the invention at 45 ° C.
FIG. 2 (a) shows the spectral peaks obtained by incubating E. coli cells expressing luciferase enzyme in citrate buffer with D-luciferin. The enzyme used was recombinant wild-type Photinus pyralis luciferase.
FIG. 2 (b) shows the spectral peaks obtained by incubating E. coli cells expressing the luciferase enzyme in citrate buffer with D-luciferin. The enzyme used is the D357K mutant.
FIG. 2 (c) shows the peak of the spectrum obtained by incubating E. coli cells expressing the luciferase enzyme in citrate buffer with D-luciferin. The enzyme used is a D357N mutant.
FIG. 2 (d) shows the spectral peaks obtained by incubating E. coli cells expressing the luciferase enzyme in citrate buffer with D-luciferin. The enzyme used is the D357W mutant.
FIG. 2 (e) shows the spectral peaks obtained by incubating E. coli cells expressing the luciferase enzyme in citrate buffer with D-luciferin. The enzyme used is the D357I mutant.
FIG. 2 (f) shows the spectral peaks obtained by incubating E. coli cells expressing luciferase enzyme in citrate buffer with D-luciferin. The enzyme used is the D357F mutant.
FIG. 2 (g) shows the spectral peaks obtained by incubating E. coli cells expressing the luciferase enzyme in citrate buffer with D-luciferin. The enzyme used is the D357Y mutant.
FIG. 2 (h) shows the spectral peaks obtained by incubating E. coli cells expressing luciferase enzyme in citrate buffer with D-luciferin. The enzyme used is the double mutant E354I + D357Y.
FIG. 3 is a graph showing% residual activity versus time for three mutant enzymes E354I, D357Y and double mutant (DM) E354I / D357Y.
FIG. 4 (a) shows an emission spectrum of the recombinant wild-type enzyme.
FIG. 4 (b) shows an emission spectrum of double mutant type (DM) E354I / D357Y.
FIG. 5 is a graph showing rate decay of photon luminescence of recombinant wild type (♦) r-wt and D357K mutant enzyme (■).
FIG. 6 shows a model diagram of the molecule showing possible CoA binding pockets in the luciferase enzyme.
FIG. 7a: Mutant P. cerevisiae grown on LB. Figure 2 shows the in vivo bioluminescence spectrum emitted by E. coli cells expressing Pyralis luciferase D357Y.
FIG. 7b: Mutant P. mori grown on minimal medium and sodium acetate. Figure 2 shows the in vivo bioluminescence spectrum emitted by E. coli cells expressing Pyralis luciferase D357Y.
FIG. 7c: Mutant P. mori grown on minimal medium and glucose. Figure 2 shows the in vivo bioluminescence spectrum emitted by E. coli cells expressing Pyralis luciferase D357Y.
FIG. 8 (a) Mutant P. groWth grown on LB. Figure 2 shows in vivo bioluminescence spectra emitted by E. coli cells expressing Pyralis luciferase E354K / D357M.
FIG. 8 (b): Mutant P. mori grown on minimal medium and sodium acetate. Figure 2 shows in vivo bioluminescence spectra emitted by E. coli cells expressing Pyralis luciferase E354K / D357M.
FIG. 8 (c). Mutant P. mori grown on minimal medium and glucose. Figure 2 shows in vivo bioluminescence spectra emitted by E. coli cells expressing Pyralis luciferase E354K / D357M.
FIG. 9 FIG. 5 is a graph showing the effect of CoA on the spectral distribution of light emitted by Pyralis luciferase D357Y.
FIG. 10 FIG. 5 is a graph showing the effect of CoA on the spectral distribution of light emitted by Pyralis luciferase D357Y.
FIG. 11 (a). FIG. 6 is a graph showing the effect of CoA on the spectral distribution of light emitted by Pyralis luciferase E354I / D357Y.
FIG. 11 (b). FIG. 6 is a graph showing the effect of CoA on the spectral distribution of light emitted by Pyralis luciferase E354I / D357Y and normalized data.
FIG. 12 shows the restriction site modifications used in the construction of the synthetic luciferase gene.
FIG. 13 shows the construct used in the synthesis of the luciferase gene.
FIG. 14: cDNA sequence of the synthetic luciferase gene (SEQ ID NO: 1) (forms part of the ribosome binding site and includes non-coding nucleotides 1-8) and encoding starting with the methionine residue indicated by the up arrow The amino acid sequence (SEQ ID NO: 2) is shown.
FIG. 15 shows the thermal stability at 50 ° C. of mutants, including mutants encoded by synthetic genes.
[Sequence Listing]

Claims (17)

Photinus pyralis, Photuris pennsylvanica, Luciola mingrelica , Luciola cruciata, Luciola lateralis, Hotaria parvula, Pyrophorus plagiophthalamus, Lampyris noctiluca, a recombinant protein with a Pyrocoelia miyako or luciferase activity derived from wild-type luciferase from Phrixothrix,, wild-type The amino acid corresponding to residue 357 of Photinus pyralis luciferase is mutated to an amino acid selected from the group consisting of tyrosine, phenylalanine, tryptophan, asparagine, glutamine, serine and threonine, and as a result, compared with the corresponding wild-type luciferase The recombinant protein can emit light of a different wavelength and / or has improved thermostability compared to the corresponding wild-type luciferase . The wild-type luciferase is, Photinus pyralis, Luciola mingrelica, a luciferase derived from Luciola cruciata or Luciola lateralis, the recombinant protein of claim 1.   The recombinant protein according to claim 1, wherein the amino acid residue corresponding to residue 357 in Photinus pyralis luciferase is mutated to tyrosine. A recombinant protein having a mutation in at least one of the following amino acid residues as compared to wild-type luciferase:
(A) Photinus pyralis luciferase amino acid residue corresponding to residue 356 of residues 354 or Luciola in luciferase;
(B) amino acid residue corresponding to residue position 217 of Photinus pyralis residue 215 of or Luciola in luciferase in luciferase;
(C) Photinus pyralis residues in luciferase 214 or Luciola mingrelica,, amino acid residue corresponding to residue 216 of Luciola cruciata or Luciola lateralis luciferase;
(D) Photinus pyralis residues in luciferase 232 or Luciola mingrelica,, amino acid residue corresponding to residue 234 of Luciola cruciata or Luciola lateralis luciferase;
(E) Photinus pyralis residues in luciferase 295 or Luciola mingrelica,, amino acid residue corresponding to residue 297 of Luciola cruciata or Luciola lateralis luciferase;
(F) an amino acid residue corresponding to residue 14 in Photinus pyralis luciferase, or residue 16 of Luciola mingrelica luciferase , or residue 17 of Luciola cruciata or Luciola lateralis luciferase ;
(G) an amino acid residue corresponding to residue 35 in Photinus pyralis luciferase, or residue 37 of Luciola mingrelica luciferase , or residue 38 of Luciola cruciata or Luciola lateralis luciferase ;
(H) an amino acid residue corresponding to residue 105 of Photinus pyralis luciferase, or residue 106 of Luciola mingrelica luciferase , residue 107 of Luciola cruciata or Luciola lateralis luciferase;
(I) Photinus pyralis luciferase residues 234 or Luciola mingrelica,, amino acid residue corresponding to residue 236 of Luciola cruciata or Luciola lateralis luciferase;
(J) an amino acid residue corresponding to residue 420 of Photinus pyralis luciferase or residue 422 of Luciola mingrelica, Luciola cruciata or Luciola lateralis luciferase ;
(K) an amino acid residue corresponding to residue 310 of Photinus pyralis luciferase or residue 312 of Luciola mingrelica, Luciola cruciata or Luciola lateralis luciferase ;
4. The recombinant protein according to any one of claims 1 to 3, characterized in that the recombinant protein has an increased thermal stability compared to an enzyme having an amino acid at this position of the corresponding wild type luciferase. Recombinant protein.   The recombinant protein according to any one of claims 1 to 4, wherein an amino acid residue corresponding to residue 354 of Photinus pyralis luciferase or residue 356 in Luciola luciferase is mutated. Photinus pyralis residues in luciferase 214 or Luciola mingrelica,, Luciola cruciata or Luciola amino acid residue corresponding to residue 216 of luciferase lateralis is, isoleucine, leucine, valine, methionine, tryptophan, phenylalanine, proline, cysteine Contact and alanine 6. The recombinant protein according to claim 5, which is mutated to a different hydrophobic amino acid selected from the group consisting of. The amino acid residue corresponding to residue 214 in Photinus pyralis luciferase or residue 216 of Luciola mingrelica, Luciola cruciata or Luciola lateralis luciferase is mutated to a different hydrophobic amino acid selected from the group consisting of isoleucine, leucine and valine The recombinant protein according to claim 6.   A nucleic acid encoding the recombinant protein according to any one of claims 1 to 7.   The nucleic acid according to claim 8, comprising a synthetic gene.   9. The nucleic acid of claim 8, wherein codon usage is optimized for a particular expression host and / or a unique restriction site is introduced.   The nucleic acid according to claim 8 or 9, comprising nucleotides 9 to 1661 of SEQ ID NO: 1.   A vector comprising the nucleic acid according to any one of claims 8 to 11.   A cell transformed with the vector according to claim 12.   A method for producing the recombinant protein according to any one of claims 1 to 7, comprising the cultivation of the cell according to claim 13.   Use of the recombinant protein according to any one of claims 1 to 7 in a bioluminescence assay.   A kit comprising the recombinant protein according to any one of claims 1 to 7.   The kit according to claim 16, further comprising luciferin.

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